UNIVERSIDADE DE SÃO PAULO

INSTITUTO DE FÍSICA DE SÃO CARLOS

VALERIA SPOLON MARANGONI

Theranostic nanomaterials applied to the cancer diagnostic and therapy and

nanotoxicology studies

São Carlos

2016

VALERIA SPOLON MARANGONI

Theranostic nanomaterials applied to the cancer diagnostic and therapy and

nanotoxicology studies

Corrected version (Original version available on the Program Unit)

São Carlos

2016

Thesis presented to the Graduate Program in

Physics at the Instituto de Física de São

Carlos, Universidade de São Paulo to obtain

the degree of Doctor of Science.

Concentration area: Applied Physics

Option: Biomolecular Physics

Advisor: Prof. Dr. Valtencir Zucolotto

To my parents, Marcia e Adenir,

and my brother Bruno

for their love and support

ACKNOWLEDGEMENTS

I would like to express my deep gratitude to all people and institutions that have

contributed direct or indirectly during my graduate school. First, I would like to acknowledge

God for all opportunities to learn and be better and to keep me strong to overcome all

adversities.

I deeply acknowledge my parents Marcia and Adenir for always provide education for

me and my brother. I thank them for being my friends, encouraging me in all my decisions,

and for their unconditional love and patience.

I deeply acknowledge Pedro for being “more than just a partner..”, for being my best

friend. I also thank his family, in special his parents Rosa Helena and Adalmir, for making me

feel as family and always encouraging me.

I deeply acknowledge my advisor prof. Dr. Valtencir Zucolotto for all opportunities

and trust during these years. I highly appreciate the freedom I had on choosing my projects

and pursuing them. I thank him for always involve me in exciting projects, which have

expanded my knowledge.

I deeply acknowledge prof. Dr. Naomi Halas for receiving me twice in your laboratory

during the PhD. I thank her for trust me and give me all support to develop the project. I

highly appreciate her suggestions and constructive criticism that have made me a better

researcher and person.

I deeply thank Dr. Oara Neumann for being not only a researcher partner, but, more

importantly, a good friend. I thank her for supporting me in the lab, presentations, writing, for

walking me home and give me many advices. I have no words to express all my gratitude.

I deeply acknowledge prof. Dr. Jim Bankson. Specially, I thank Caterina Kaffes for

helping with the MRI measurements and for always being willing to stay during the nights

and weekends doing the measurements with me.

I thank prof. Dr. Peter Nordlander and his student Hui Zhang for helping with the

theoretical model for the MRI-active nanoparticles, which allowed a better understanding

about the system.

I deeply acknowledge Dr. Sandra Bishnoi for helping me in writing the project and

supporting me in the research. I highly appreciate her help (inside and outside the Lab) and

the helpful discussions.

I deeply acknowledge Dr. Ciceron Ayala-Orozco, my mentor during the beginning of

my internship in Houston, for guiding me quickly throughout the synthesis.

I deeply thank to my dear friend and research collaborator, Dra. Juliana Cancino-

Bernardi for helping me in the experiments, especially those of tens of cell culture plates.

I thank Dra. Lilian Centurion for being a good friend and collaborator during all these

years. I also thank her for helping in the text revision of this thesis.

I thank everybody that keeps the GNano lab working: Jaque, Julielle, Romeu, Nelzeli.

I thank Martha Alexander, Sandy Willians, Surbhi Lal, for all of the work with the invoices at

LANP (Rice University) and for helping me with the visa paperwork.

I thank my very good friends from GNano: Lais Brazaca, Jaque, Camilo, Idelma,

Nirton, Lorena, Paula, Henrique, Fran, Cris, Lais Ribovisk, Helena, Adrislaine, Isa, Olavo,

Abilene. I also thank Ila and Clara for helpful discussions in the cell culture room and

friendship.

I am also grateful to my close friends Paty, Ana Eliza, Amanda and our princess Sofia.

I thank them for their help and support all the time.

I thank Nayára Oliveira to show me the importance of the self-knowledge for the

personal and professional evolution during the coaching program.

I thank all the undergrad and masters students that I have opportunity to guide during

all these years. The opportunities to work with different projects and problems have made me

a better researcher and person.

I thank my dear friend Tatiana Wolfe for helping me in Houston, especially in the first

time in 2012. I highly appreciate her friendship, support, and scientific discussions. I also

would like to thank my good Brazilian friends in Houston: Caio, Angelica, Daniel, Fabio,

Gustavo, Leonardo.

I deeply thank my very good friends in Houston: Pratiksha, Tiyash, Mihika,

Liangliang. Thank you for supporting and helping me in the difficult moments and for

showing me different cultures and foods.

I thank all Halas’ group members, especially Andie, Adam, Ali, Dayne, Alejandra,

Ben, Bob, Linan, Chao, Fanfang, Shu, Amanda, Michael, Nate, Sam for all useful discussions

and help.

I deeply acknowledge Instituto de Física de São Carlos - IFSC, for the award “Yvonne

Primerano Mascarenhas", which give me the financial support to do my first internship at

Rice University in 2012.

I thank Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) for the

fellowship during the PhD (2012/11166-4) and for the research internships abroad (BEPE)

(2014/13645-2). I also would like to thank the opportunities to participate of international

conferences, which have expanded my vision of science and world.

I acknowledge the Laboratorio de Microscopia Eletrônica (Laboratório Nacional de

Nanotecnologia – LNNano, Campinas) for the training and possibility to use the Transmission

Electron Microscopy many times during the PhD.

I thank the IFSC multi-user laboratories: Laboratorio de Microscopia Eletronica and

Microscopia confocal. I would like to thank prof. Dr Francisco Guimarães for helping with

the confocal microscopy and Daniela Correa de Melo for helping with the Iatroscan

measurements.

I deeply acknowledge the post-graduation office and library for always being willing

to help the students, especially with the thesis and deadlines.

I thank Rice University for providing a singular infrastructure and cultural activities

during my internship program. I deeply thank Dr. Pennington and Dr. Wenhua for training me

on ICP-MS and TEM, and Dr. Budi Utama for helping with the confocal measurements. I

thank the faculty and staff of Chemistry Department, the Office of International Students and

Scholars (OISS), and of the collaborating institution MD Anderson Cancer Center. I thank

Mayra Onuchic for the important work at Brazil@Rice, and all support.

Finally, I would like to thank Universidade de Sao Paulo, especially Instituto de

Química de Sao Carlos (IQSC) and Fisica de Sao Carlos (IFSC), and all professors and

faculty, for the exceptional education that I received in this decade (bachelor, master and

PhD).

“In a gentle way, you can shake the world”

Mahatma Gandhi

ABSTRACT

MARANGONI, V. S. Theranostic nanomaterials applied to the cancer diagnostic and

therapy and nanotoxicity studies. 2016. 154 p. Thesis (Doctor in Science) - Instituto de

Física de São Carlos, Universidade de São Paulo, São Carlos, 2016.

Multifunctional plasmonic nanoparticles have shown extraordinary potential for near infrared

photothermal and triggered-therapeutic release treatments of solid tumors. However, the

accumulation rate of the nanoparticles in the target tissue, which depends on their capacity to

escape the immune system, and the ability to efficiently and accurately track these particles in

vivo are still limited. To address these challenges, we have created two different systems. The

first one is a multifunctional nanocarrier in which PEG-coated gold nanorods were grouped

into natural cell membrane vesicles from lung cancer cell membranes (A549) and loaded with

β-lap (CM-β-lap-PEG-AuNRs). Our goal was to develop specific multifunctional systems for

cancer treatment by using the antigens and the unique properties of the cancer cell membrane

combined with photothermal properties of AuNRs and anticancer activity of β-lap. The results

confirmed the assembly of PEG-AuNRs inside the vesicles and the irradiation with NIR laser

led to disruption of the vesicles and release of the PEG-AuNRs and -Lap. In vitro studies

revealed an enhanced and synergic cytotoxicity against A549 cancer cells, which can be

attributed to the specific cytotoxicity of -Lap combined with heat generated by laser

irradiation of the AuNRs. No cytotoxicity was observed in absence of laser irradiation. In the

second system, MRI-active Au nanomatryoshkas were developed. These are Au core-silica

layer-Au shell nanoparticles, where Gd(III) ions are encapsulated within the silica layer

between the inner core and outer Au layer of the nanoparticle (Gd-NM). This theranostic

nanoparticle retains its strong near infrared optical absorption properties, essential for in vivo

photothermal cancer therapy, while simultaneously providing increased T1 contrast in MR

imaging by concentrating Gd(III) within the nanoparticle. Measurements of Gd-NM revealed

a substantially enhanced T1 relaxivity (r1 ~ 17 mM-1

s-1

) even at 4.7 T, surpassing

conventional Gd(III)-DOTA chelating agents (r1 ~ 4 mM-1

s-1

) currently in clinical use. The

observed relaxivities are consistent with Solomon-Bloembergen-Morgan (SBM) theory,

describing the longer-range interactions between the Gd(III) and protons outside the

nanoparticle. These novel multifunctional systems open the door for the development of more

efficient nanoplatforms for diagnosis and treatment of cancer.

Keywords: Plasmonic nanoparticles. Photothermal therapy. Magnetic resonance imaging.

RESUMO

MARANGONI, V. S. Nanomateriais Teranósticos Aplicados à Problemática do Câncer e

Estudos de Nanotoxicidade. 2016. 154 p. Tese (Doutorado em Ciências) - Instituto de Física

de São Carlos, Universidade de São Paulo, São Carlos, 2016.

Nanopartículas plasmônicas multifuncionais têm revelado elevado potencial para fototermia

na região (NIR) do infravermelho e liberação controlada de fármacos para o tratamento de

tumores sólidos. No entanto, a taxa de acumulação das nanoparticulas no tecido alvo, que

depende da capacidade delas de escapar do sistema imunológico, e a habilidade de rastrear de

maneira efetiva essas partículas in vivo ainda são limitadas. Para superar essas barreiras, dois

sistemas diferentes foram desenvolvidos. O primeiro corresponde a um nanocarreador

multifunctional, onde nanobastões de ouro funcionalizados com PEG foram agrupados dentro

de vesículas de membranas de células naturais originarias de células cancerígenas de pulmão

(A549) conjugadas com β-Lap (CM-β-lap-PEG-AuNRs). Nosso principal objetivo foi

desenvolver um sistema multifuncional especifico para tratamento de câncer utilizando os

antígenos e propriedades únicas da membrana das células cancerígenas combinados com as

propriedades fototérmicas dos AuNRs e a atividade anticancerígena da β-Lap. Os resultados

confirmaram o agrupamento dos PEG-AuNRs dentro das CM e irradiação com o laser no NIR

levou ao rompimento das vesículas e liberação dos AuNRs e -Lap. Estudos in vitro

revelaram uma elevada e sinérgica citotoxicidade contra células A549, que pode ser atribuída

a combinação da especifica toxicidade da -Lap com o calor gerado pelos AuNRs por meio

da irradiação com laser. Nenhuma citotoxicidade significativa foi observada na ausência de

irradiação com laser. No segundo sistema, nanomatryoshkas de Au ativas em MRI foram

desenvolvidas. Elas consistem em um núcleo de Au, uma camada intersticial de sílica, onde

os íons de Gd(III) são encapsulados, e uma camada externa de Au (Gd-NM). Esta

nanopartícula teranóstica mantém as propriedades de elevada absorção óptica no NIR,

enquanto simultaneamente fornece um elevado contraste T1 em imagem por ressonância

magnética por meio da concentração dos íons de Gd(III) dentro da nanoparticula. Medidas de

Gd-NM revelaram uma relaxividade elevada (r1 ~ 17 mM-1

s-1

) a 4,7 T, superando os

convencionais agentes quelantes de Gd(III)-DOTA (r1 ~ 4 mM-1

s-1

) utilizados clinicamente.

As relaxividades observadas são consistentes com a teoria Solomon-Bloembergen-Morgan

(SBM), descrevendo as interações de longo alcance entre Gd(III) e prótons de H fora da

partícula. Os novos sistemas multifuncionais desenvolvidos abrem oportunidades para o

desenvolvimento de nanoplataformas mais eficientes para o diagnóstico e tratamento de

câncer.

Palavras-chave: Nanopartículas plasmônicas. Terapia fototérmica. Imagem por ressonância

magnética.

LIST OF FIGURES

Figure 2.1 - Schematic representation of the SPR oscillation and its respective

extinction spectra for gold (A) nanospheres and (B) nanorods. ........................ 39

Figure 2.2 - Schematic representation of a gold nanoshell and a representative

extinction spectrum for a [r1, r2] = [62, 76] nm.................................................. 42

Figure 2.3 - Schematic energy-level diagram describing the plasmon hybridization in

gold nanoshells resulting from the interaction between the sphere and

cavity plasmons .................................................................................................. 43

Figure 2.4 - Schematic representation of a gold nanomatryoshka and a representative

extinction spectrum for a [r1, r2, r3] = [24, 34, 44] nm. ...................................... 44

Figure 2.5 - Schematic representation of the gold nanorod synthesis process. (A) First,

small gold nanoparticles are synthetized in the presence of CTAB, and

(B) used as seeds for the growth of the nanorods. ............................................. 49

Figure 2.6 - Characterization of the seeds: (A) UV-VIS-NIR spectrum, (B)

Hydrodynamic diameter obtained from Dynamic Light Scattering

measurements, and (C) TEM image, showing very small particles. .................. 49

Figure 2.7 - (A) UV-VIS-NIR spectra showing the growth kinetics of nanorods after

addition of 100 µL of seeds into the growth solution, and (B) the changing

in the maximal wavelength of the longitudinal band as function of time. ......... 50

Figure 2.8 - UV-VIS-NIR spectra of AuNRs obtained using different amount of AgNOs

and the respective TEM images. The aspect ratio are 2.1, 2.8, and 3.5 for

blue, green, and red, respectively. Scale bar = 50 nm. ....................................... 51

Figure 2.9 - TEM images of the gold nanorods with maximal longitudinal absorption at

700 nm, showing a monodisperse system. ......................................................... 51

Figure 2.10 - UV-VIS-NIR spectra of gold nanorods prepared with different

concentrations of seeds. ..................................................................................... 52

Figure 2.11 - (A) Schematic representation of the gold nanomatryoshka synthesis: 50

nm diameter gold colloids are coated with SiO2-APTES shell, followed by

a continuous gold shell. ...................................................................................... 53

Figure 2.12 - UV-VIS-NIR extinction spectra and their respective TEM images

corresponding to (A) Au colloids of about 50 nm, (B) APTES-SiO2-

coated Au colloids, and (C) gold nanomatryoskas. ........................................... 54

Figure 2.13 - UV-VIS-NIR spectra of gold nanomatryoskas obtained using different

amount of seeded precursor. ............................................................................... 55

Figure 2.14 - UV-VIS-NIR spectrum of Au-coated Au2S nanoshells and its respective

TEM image. ........................................................................................................ 56

Figure 2.15 - Analysis of the composition of the (A) nanoshells and (B) triangular

plates by High Resolution TEM coupled with energy dispersive

spectroscopy (EDS). ........................................................................................... 56

Figure 2.16 - Pictures of the centrifuge tubes before and after the centrifugation

showing the separation of the particles through the density gradient, and

the UV-VIS-NIR spectra and TEM images of the two indicated fractions. ....... 57

Figure 2.17 - UV-VIS-NIR spectra and their respective TEM images of different

synthesis process to synthesized Au-coated Au2S nanoshells. (A)

Na2S2O3:HAuCl4 (18 mL:10 mL); (B) Na2S : HAuCl4 (18 mL : 10 mL)

under N2 atmosphere; (C) Na2S : HAuCl4 (18 mL : 10 mL) in pH = 1.6,

and (D) Na2S : HAuCl4 (10 mL : 10 mL), and after 5 min, more 2 mL of

Na2S were added. ............................................................................................... 58

Figure 2.18 - UV-VIS-NIR spectrum and the respective TEM image taken 1h30 after

mixing HAuCl4 with NaOH and then with Na2S. .............................................. 59

Figure 2.19 - UV-VIS-NIR spectrum and the respective TEM image of the Au-coated

Au2S nanoshells prepared by a new two-step method........................................ 60

Figure 3.1 - The near infrared region (NIR) window for in vivo applications, showing

the region of minimal light absorption by hemoglobin and water. .................... 65

Figure 3.2 - Schematics of the synthesis of the Red blood cell (RBC) membrane-coated

polymeric PLGA nanoparticles. ......................................................................... 69

Figure 3.3 - Chemical structure of -Lapachone. .................................................................... 71

Figure 3.4 - Schematic representation (not to scale) of proposed mechanism for the

fabrication of the cancer cell membrane-coated PEG-Gold nanorods and

loaded with the anticancer agent -Lapachone (CM--Lap-PEG-AuNRs).

First, A549 lung cancer cells are isolated, lysed to remove the internal

content and purified by ultracentrifugation. -Lap is loaded on cell

membrane structure and they are extruded through 200 porous membrane

(CM--Lap). In parallel, gold nanorods (AuNRs) are synthesized by the

seed-mediated method in presence of surfactant CTAB, followed by

covalent functionalization with mPEG-SH. The fusion of the resulting

PEG-AuNRs and CM--Lap are performed by mechanical extrusion

through 100 nm pore membrane. ....................................................................... 83

Figure 3.5 - a) Representative spectra of -Lap in water:ethanol (1:1) in different

concentrations and b) Calibration curve of -Lap at 257 nm made from

triplicate of the spectra in different concentrations. ........................................... 83

Figure 3.6 - Calibration curve of BSA protein obtained from bicinchoninic acid (BCA)

assay and used to determine the protein concentration on the cell

membranes vesicles (CM). ................................................................................. 84

Figure 3.7 - Characterization of the A549 cell membrane (CM). (A) Polyacrylamide

gel electrophoresis (PAGE-SDS) of the CM in duplicate. (B) Dynamic

light scattering analysis of the CM--Lap after extrusion extrusion

through 200 nm porous polycarbonate membrane, and (C) UV-VIS

spectra of CM with/without -Lapachone. ........................................................ 85

Figure 3.8 - Iatroscan TLC/FID chromatograms of standard lipids and A549 cell

membrane (CM) samples. .................................................................................. 86

Figure 3.9 - (A) Representative FEG-SEM image of the PEG-AuNRs and (B)

Histogram of length and (C) width determined by measuring about 100

particles using the software ImageJ. The average aspect ratio

(length/width) calculated from the mean 47.4/13.37 was about 3.5. ................. 87

Figure 3.10 - Characterization of gold nanorods coated with cell membrane loaded -

Lapachone (CM--Lap-PEG-AUNRs) and comparison with PEG-AuNRs.

(A) UV-VIS-NIR spectra and (B) hydrodynamic diameter obtained from

DLS measurements of PEG-AuNRs before and after coating with CM--

Lap. Longitudinal plasma resonance of PEG-AuNRs (λmax~750 nm)

decreased after the coating. C) Zeta potential of the AuNRs before and

after functionalizations, D) stability of CM--Lap-PEG-AuNRs in PBS

buffer and 10% Fetal Bovine Serum; (e) and (f) FEG-SEM images of

CM--Lap-PEG-AuNRs showing the PEG-AUNRs grouped inside the

cell membrane vesicles. ..................................................................................... 89

Figure 3.11 - (A) Representative FEG-SEM image of CM--PEG-AuNRs and (B)

histogram determined by measuring about 100 particles using the

software ImageJ. The average diameter calculated was about 109 nm. ........... 90

Figure 3.12 - Representative FEG-SEM image of the CM--Lap-coated CTAB-AuNRs.

No spherical cell membrane vesicles are observed, suggesting their

destabilization in presence of the surfactant CTAB. .......................................... 90

Figure 3.13 - Plots of heat flow vs time of microcalorimetric titration of (a) PEG-

AuNRs; (b) CM--Lap-PEG-AuNRs and (c) CTAB/PEG-AuNRs by BSA

solution. Injections of 2 μL, at 25 °C, of 0.01 mM of BSA solution were

made on nanoparticles suspensions. Control of BSA dilution was carried

out by injections of BSA solution in buffer. ...................................................... 92

Figure 3.14 - (A) Representative of temperature change vs time in suspensions (optical

density at 750 nm = 1) irradiated with 808 nm laser with power density of

1.5 W/cm2, (B) Increase of the longitudinal band with the laser

irradiation, (C) release of -Lapachone at different time of irradiation.

SEM images of CM--Lap-PEG-AuNRs (D) before, (E) after 10 min and

(F) after 20 min of laser irradiation. The laser irradiation increases the

temperature of the suspension, breaks the vesicles and causes the release

of the AuNRs ...................................................................................................... 94

Figure 3.15 - Photothermal therapy of A549 cancer cells treated with CM--Lap-PEG-

AuNRs and CM-PEG-AuNRs. The highlight areas represent

approximately the laser spots (~1.5 W/cm2, 10 min) on the samples. Dead

cells were stained with Trypan Blue. Cells without the particles are not

affected by the laser irradiation while cells incubated with CM--Lap-

PEG-AuNRs and CM-PEG-AuNRs were visibly injured. ................................. 95

Figure 3.16 - Photothermal therapy of A549 cancer cells treated with CM--Lap-PEG-

AuNRs and CM-PEG-AuNRs for 4 h. The highlight areas represent

approximately the laser spots (~1.5 W/cm2, 10 min) on the samples. Dead

cells were stained with Trypan Blue. Cells without the particles are not

affected by the laser irradiation while cells incubated with CM--Lap-

PEG-AuNRs and CM-PEG-AuNRs were visibly injured. ................................. 96

Figure 3.17 - (A) Confocal images of A549 cells after cell exposure to CM-PEG-

AuNRs for 4 h. Blue: cell nuclei (Hoechst 33258). red: CellMask Deep

Red-labeled CM-PEG-AuNRs (Exc/Em = 649/667 nm). (B) Time

dependence of the extinction at 750 nm of the cells treated with CM--

Lap-PEG-AuNRs, showing the dynamic of the uptake of the particles.

The data were corrected by the number of cells (relative ext) and are

showed as mean ± SD of triplicate. (C) Comparison of the uptake of PEG-

AuNRs and CM--Lap-PEG-AuNRs after 4 h of incubation. The

extinctions were corrected by the number of cells and normalized. .................. 98

Figure 3.18 - Intracellular uptake of CM-PEG-AuNRs. Samples were incubated with

A549 cells for 4 h. Red: CellMask Deep Red-labeled CM-PEG-AuNRs

(Exc/Em = 649/667 nm); Blue: nuclei stained with Hoechst 33258. CM-

PEG-AuNRs show strong signal when nucleus is in focus (middle row),

demonstrating cellular uptake. ........................................................................... 99

Figure 3.19 - Confocal images of A549 cells after cell exposure to CellMask Deep Red

stain (a, b, c) and CellMask-stained CM-PEG-AuNRs (d, e, f) for 4 h.

Blue: cell nuclei (Hoechst 33258). ................................................................... 100

Figure 3.20 - Cell viability, evaluated by (a) MTT; (b) Crystal Violet, and (c) MTT of

A549, HTC and HepaRG cells after incubation with the samples for 24 h.

In (a) and (b) the columns blue, green, and red represent the quantities of

5, 10 and 15 µL, respectively. In (c) the columns red, green, blue, light

blue and pink represents the concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0

µM of -Lap, respectively. The relative percentage of viable cells was

calculated by considering the control as 100% of viable cells. The results

represent the mean ± SD of data normalized to untreated controls from

two independent experiments in triplicate. *P<0.05 with respect to

untreated controls. ............................................................................................ 102

Figure 3.21 - Measurement of generation of ROS in A549 and HepaRG cells. (a) Cells

were incubated with the samples and the generation of ROS was

measured using the 2',7'-dichlorodihydrofluorescein

diacetate (H2DCFDA) assay. The relative percentage was calculated by

considering the control as 100%. Data are expressed as mean ± SD of

triplicate from two independent experiments. *P<0.05 with respect to

untreated controls. ............................................................................................ 103

Figure 4.1 - Schematic showing the main differences between T1 and T2 relaxation. .......... 107

Figure 4.2 - (A) Schematic representation of the MRI active nanomatryoshka synthesis

showing the stepwise synthesis process: the 50 nm diameter gold colloids

are coated with Gd(III) chelates embedded in a SiO2 shell and then a

continuous Au shell. (B) TEM images corresponding to each step in the

process; scale bars are 100 nm. ........................................................................ 120

Figure 4.3 - (A) FTIR spectra of (i) Au@SiO2-APTES, (ii) Gd-DOTA-SCN, and (iii)

Gd-doped SiO2-coated Au colloid. The disappearance of the 2100 cm

-1

peak, attributed to the N=C=S vibration, is an indicative of the bond

between Gd-DOTA-SCN and the amine groups from APTES (B)the

chemical reaction showing the bond formation. The table shows the FTIR

peak assessments corresponding to each spectrum. ......................................... 121

Figure 4.4 - High resolution TEM images of: (A) SiO2-coated Au colloid and (B) Gd-

doped SiO2 coated Au colloid. ........................................................................ 121

Figure 4.5 - (A) The r1 relaxivity of Gd-NM as a function of the number of Gd(III) per

NM measured at 4.7 T. (B)The r1 relaxivities of Gd(III)-DOTA (for

comparison), Gd(III)-NM, and Gd(III)-NM-PEG; error bars represent

standard deviation of three independent experiments at 25ºC. (C)

Thermal variation of T1 for Gd-NM-PEG at 25ºC (blue, r1 = 14.6 mM-1

s-

1) and 37ºC (red, r1 = 16.1 mM

-1 s

-1). The r1 values were extracted from

the slopes. ......................................................................................................... 122

Figure 4.6 - T1 (longitudinal) rate of Gd-NM for: (A) 0.8x10

5

, (B) 2.7x105

, and (C)

8.2x105

numbers of Gd(III) ions per particle at 4.7 T and 25ºC. The r1

values were extracted from the slopes. ............................................................. 123

Figure 4.7 - (A) Dynamic Light Scattering and (B) Zeta potential for Gd-NM before

and after PEG surface functionalization. .......................................................... 124

Figure 4.8 - (A) Schematic representation of (A) the regular NM synthesis and (B) the

functionalization of the NM with Gd(III) chelate (GdPEG-coated-NM) ......... 125

Figure 4.9 - (A) Dynamic Light Scattering and (B) Zeta potential for regular NM before

and after conjugation with PEG-NH2 and Gd chelates (GdPEG-coated-

NM). ................................................................................................................. 125

Figure 4.10 - Percentage of Gd(III) ions released from Gd-NM-PEG, where the Gd(III)

is located inside the NM which is then functionalized using PEG, versus

GdPEG-coated NM, where the Gd(III)-DOTA-SCN is attached outside

the NM through a thiol-PEG-amine linker, after 48 h of incubation at

37ºC in 10% FBS (red) and H2O (blue). Gd(III) concentration was

quantified using ICP-MS. ................................................................................. 126

Figure 4.11 - (A) T1 (longituginal) rate and (B) extinction spectra for Gd-NM-PEG in

water (blue) and 10% serum (red). (C) T1 (longituginal) rate and (D)

extinction spectra GdPEG-coated NM in water (blue) and 10% serum

(red). The T1 measurements were performed at 4.7 T and 25ºC. ..................... 127

Figure 4.12 - T1 (longitudinal) rate versus Gd(III) concentration at 4.7 T for (A) seeded

precursor, (B) Gd-NM-PEG, and (C) GdPEG-coated-NM; and the

corresponding (D,E,F) extinction spectra and (G,H,I) TEM images; (scale

bar are 50 nm). (Insets A-C: T1 weighted MR images). ................................... 129

Figure 4.13 - (A) Longitudinal rate at 4.7 T versus Gd(III) concentration for two Au

shell thicknesses of Gd-NM-PEG: 18 nm (blue; r1 = 12.9 mM-1

s-1

) and 38

nm (red; r1 = 1.2 mM-1

s-1

) the Gd(III) concentration per NM was 2.3x105

in both cases, (B) T1 weighted MR images corresponding to the

concentrations shown in (A), and corresponding TEM images. (C)

extinction spectra of both Gd-NM-PEG nanoparticles as denoted in (A),

and (D) the calculated longitudinal relaxivity r1 versus Au shell thickness

(green) using SBM theory (a concentration of 2.3x105Gd(III) chelates per

NM is used). The blue and red dots are the corresponding experimental

relaxitivities. ..................................................................................................... 132

Figure 4.14 - (A) Flow cytometry toxicity analysis using SYTOX® red dead cell stain

of RAW 264.7 cells after 24 h of incubation with Gd-NM-PEG (green)

and Gd-coated-NM (blue). The percentages were calculated considering

the control as 100%. (B) Bright field microscopy and (C) merged

confocal and reflectance images of RAW 264.7 macrophage cells

showing the nuclei (blue) and Gd-NM-PEG (red). T1-weighted MR

images of (D) reference (phosphate buffer solution (1x PBS)), (E) RAW

267.4 macrophage cells in PBS, and (F) RAW 267.4 macrophage cells

with Gd-NM-PEG in PBS. The cell concentration was ~5.2x107cells/mL. .... 134

Figure 4.15 - Dependence of MR relaxation on nanoparticle concentration and Gd(III)

concentration in phantom: T1-weighted MR images of reference (water),

agarose, and the equivalent number of Gd-NM-PEG (100 μL of solution). .... 134

LIST OF TABLES

Table 4.1 - T1 relaxivity for clinically used contrast agents at 0.47 and 1.41 T. ................... 108

Table 4.2 - T1 relaxivity of Gd(III) conjugated nanomaterials. ............................................. 111

Table 4.3 - Hydrodynamic diameter of Gd-NM-PEG and GdPEG-coated NM in water and

after dispersion in culture media (FBS 10%) measured by dynamic light

scattering (DLS). ................................................................................................. 127

LIST OF ABREVIATIONS ANS ACRONYMS

ALC aliphatic alcohol free

AMPL mobile polar lipids in ketone

APTES 3-aminopropyl)triethoxysilane

AuNRs gold nanorods

BBB blood-brain barrier

BSA bovine serum albumin

CM natural cell membrane

CTAB cetyltrimethylammonium bromide

CV crystal violet

DIC differential interference contrast

DLS dynamic light scattering

DMEM Dulbecco’s Modified Essential Media

DTPA Gd(III)-diethylenetriamine pentaacetic acid

EDS energy dispersive spectroscopy

EPR enhanced permeability and retention

FBS fetal bovine serum

FEG-SEM field-emission Scanning Electron Microscopy

FFA free fatty acids

FID flame ionization detection

FTIR Fourier transform infrared spectroscopy

Gd-DOTA-SCN S-2(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-

1,4,7,10-tetraaceticacid

HC aliphatic hydrocarbons

ICP-MS inductively coupled plasma mass spectroscopy

MRI magnetic resonance imaging

MTT 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide

NIR near infrared region

NM Au nanomatryoshkas

NQO1 NADP(H):quinine oxidoreductase

NSF nephrogenic system fibrosis

PBS phosphate buffer saline

PDT phodynamic therapy

PEG polyethylene glycol

PL phospholipids

PLGA poly(lactic-co-glycolic acid)

PPPT plasmonic photothermal therapy

RBC red blood cell membrane

RF radiofrequency

SBM Solomon-Bloembergen-Morgan

SDS-PAGE polyacrylamide gel electrophoresis

SERS surface enhanced raman scattering

SPR surface plasmon resonance

ST sterol

TAG triglycerides

TEM transmission electron microscopy

TEOS tetraethoxysilane

THPC tetrakis(hydoxymethyl) phosphonium chloride

TLC thin-layer chromatography

UV-VIS-NIR ultraviolet-visivel-near infrared

WE/SE ester

β-Lap β-Lapachone

LIST OF SYMBOLS

dielectric constant for the metal

m dielectric constant of the surrounding medium

intrinsic relaxivity

r1 longitudinal relaxivity

r2 transverse relaxivity

inner-sphere contributions to the total relaxivity

outer-sphere contributions to the total relaxivity

proton Larmor angular frequencies

electronic Larmor angular frequencies

A experimental extinction in arbitrary units

a length of the nanoparticle

b,c width of the particle

B0 external magnetic field

C constant

Cabs cross-sections of absorption

Cext total extinction

Csca cross-sections of scattering

d distance of closest approach of H2O molecules

D sum of diffusion coefficients of bulk water and of the complex

spectral density function

l angular moment

l optical path of the cuvette in cm

M net magnetization

Mxy transverse magnetization

Mz longitudinal magnetization

N concentration of particles (number of particles/mL)

R aspect ratio (a/b)

T1 longitudinal relaxation time

T2 transverse relaxation time

V unit of volume of the nanoparticle

γ gyromagnetic ratio

λ wavelength of light

ω0 angular frequency

ωB plasmon frequency of the bulk metal

ωc,l plasmon frequency of the cavity

ωs,l plasmon frequency of the gold sphere

CONTENTS

1 INTRODUCTION AND OBJECTIVES ................................................................... 33

2 SYNTHESIS AND CHARACTERIZATION OF PLASMONIC

NANOPARTICLES .................................................................................................... 37

2.1 INTRODUCTION ...................................................................................................... 37

2.1.1 Plasmonic nanoparticles ....................................................................................................... 37

2.1.2 Gold nanorods ........................................................................................................................ 39

2.1.3 Gold nanoshells and nanomatryoshkas ............................................................................... 41

2.2 EXPERIMENTAL SECTION ...................................................................................... 45

2.2.1 Materials ................................................................................................................................. 45

2.2.2 Gold nanorods (AuNRs) synthesis ...................................................................................... 45

2.2.3 Gold nanomatryoskas (NM) synthesis ................................................................................ 46

2.2.4 Au-coated Au2S nanoshells synthesis ................................................................................. 47

2.2.5 Characterization Techniques ................................................................................................ 48

2.3 RESULTS AND DISCUSSION ................................................................................... 48

2.3.1 Gold nanorods synthesis and characterization ................................................................... 48

2.3.3 Au-coated Au2S nanoshells .................................................................................................. 55

2.4 CONCLUSIONS .......................................................................................................... 60

3 PLASMONIC GOLD NANORODS COATED WITH CANCER CELL

MEMBRANE AS A MULTIFUNCTIONAL SYSTEM FOR CANCER

THERAPY ................................................................................................................... 63

3.1 INTRODUCTION ........................................................................................................ 63

3.1.1 Theranostic nanoparticles ..................................................................................................... 63

3.1.2 Plasmonic Photothermal therapy (PPTT) ........................................................................... 64

3.1.3 Combined therapies ............................................................................................................... 67

3.1.4 Cell membrane coated nanomaterials ................................................................................. 68

3.1.5 Cell membrane vesicles as carriers ..................................................................................... 70

3.1.6 -Lapachone ........................................................................................................................... 71

3.1.7 Toxicity of nanomaterials ..................................................................................................... 72

3.2 EXPERIMENTAL SECTION................................................................................... 73

3.2.1 Materials ................................................................................................................................. 73

3.2.2 Gold nanorods (AuNRs) synthesis ...................................................................................... 74

3.2.3 Cell culture ............................................................................................................................. 74

3.2.4 Extraction of A549 cancer cell membrane (CM) ............................................................... 75

3.2.5 A549 cancer cell membrane (CM) characterization .......................................................... 75

3.2.6 A549 cell membrane-loaded β-lapachone (CM-β-Lap) .................................................... 76

3.2.7 A549 cell membrane-loaded β-lapachone-coated PEG functionalized gold nanorods

(CM-β-Lap-PEG-AuNRs) .................................................................................................... 77

3.2.8 Photothermal therapy ............................................................................................................. 77

3.2.9 Characterization ...................................................................................................................... 78

3.2.10 Isothermal Titration Calorimetry (ITC) ............................................................................... 79

3.2.11 Cellular Uptake ....................................................................................................................... 80

3.2.12 Cytotoxicity ............................................................................................................................. 81

3.2.13 Detection of Intracellular Reactive Oxygen Species (ROS) ............................................. 82

3.3 RESULTS AND DISCUSSION ....................................................................................82

3.3.1 CM characterization ............................................................................................................... 84

3.3.2 Synthesis of CM--Lap-PEG-AuNRs.................................................................................. 86

3.3.3 Photothermal properties ......................................................................................................... 92

3.3.4 In vitro photothermal therapy ................................................................................................ 95

3.3.5 Cellular uptake ........................................................................................................................ 97

3.3.6 Cytotoxicity ........................................................................................................................... 101

3.3.7 Detection of Intracellular Reactive Oxygen Species (ROS) ........................................... 102

3.4 CONCLUSIONS ..........................................................................................................103

4 Gd(III)-ENCAPSULATING AU NANOMATRYOSKHAS: ENHANCED

T1 MAGNETIC RESONANCE CONTRAST IN A THERANOSTIC

NANOPARTICLE .................................................................................................... 105

4.1 INTRODUCTION .......................................................................................................105

4.1.1 Magnetic Resonance Imaging (MRI) ................................................................................. 105

4.1.2 Gd(III) based contrast agents .............................................................................................. 108

4.1.3 Nanostructured T1 MRI contrast agents ............................................................................. 109

4.2 EXPERIMENTAL SECTION .....................................................................................112

4.2.1 Materials................................................................................................................................. 112

4.2.3 Gd-embedded Au nanomatryoshkas (Gd-NM) synthesis ................................................ 112

4.2.4 Polyethylene glycol (PEG)-functionalization of Gd-NM (Gd-NM-PEG) .................... 114

4.2.5 GdPEG-coated gold nanomatryoskas (GdPEG-coated NM) synthesis ......................... 114

4.2.6 Calculation of Nanomatryoshkas Concentration .............................................................. 115

4.2.7 Characterization .................................................................................................................... 115

4.2.8 Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) .......................................... 116

4.2.9 Gd(III) release .............................................................................................................. 117

4.2.10 MRI Analysis ........................................................................................................................ 117

4.2.11 Cytotoxicity ........................................................................................................................... 117

4.2.12 Fluorescence Imaging .......................................................................................................... 118

4.2.13 Preparations of Cells for MRI ............................................................................................. 118

4.2.14 Phantom preparation for MRI ............................................................................................. 119

4.3 RESULTS AND DISCUSSION .................................................................................. 119

4.4 CONCLUSIONS ......................................................................................................... 135

5 CONCLUSIONS AND PERSPECTIVES............................................................... 137

REFERENCES .......................................................................................................... 139

33

1 INTRODUCTION AND OBJECTIVES

Developments in nanomedicine have offered new hope in fighting cancer, one of the

leading causes of death worldwide,(1) mainly in the areas of photothermal and gene therapy,

drug delivery, and diagnostic imaging.(2-5) In particular, plasmonic gold nanoparticles have

received great attention as theranostic agents (that exhibit both diagnostic and therapeutic

functions) due to their unique properties, including intense near infrared optical absorption as

a result of their strong localized surface plasmon resonance (SPR), in vivo/in vitro stability,

biocompatibility, and facile surface conjugation chemistry.(5-6)

Plasmonic photothermal therapy (PPPT), which consists in the transduction of light by

specific nanosystems into heat, has emerged as a potential minimally invasive alternative for

cancer treatment with reduced side effects.(7) The use of long wavelength laser irradiation,

such as in the near infrared region (NIR) around 650-950 nm, is particularly advantageous

since it has the maximum radiation penetration and can be used in deeply seated targets.(8)

Gold nanorods (AuNRs) provide excellent systems for applications in PPTT due to their

localized surface plasmon resonance (SPR) at NIR.(9) Recently Halas’ group developed

tunable plasmonic Au nanomatryoshkas (NM), a metal-based nanoparticle consisting of an

Au core, an interstitial nanoscale SiO2 layer, and an outer Au shell.(10-11) This nanoparticle

possesses a strong optical extinction at 800 nm, tunable by varying its geometry if needed,

which makes it a highly attractive candidate for photothermal cancer therapy.

However, despite many studies in this area, significant clinical advances have not been

observed. One of the main reasons for this failure is the low accumulation rate of the

nanoparticles in the target tissue, which depends on their ability to escape the immune system,

cross the biological barriers and accumulate at target tissues.(12) Moreover, the ability to

determine the precise anatomical location of theranostic nanoparticles in the body, in real

time, before, during, and after treatment is important to guarantee their efficiency and

safety.(13)

The functionalization of nanoparticles with polyethylene glycol (PEG) has been the

gold standard to extend the in vivo blood circulation time of the particles and to improve their

passive and active accumulation in the tumor.(14) Although much progress has been made,

the accumulation in the target region is still low, reducing the therapeutic effects of

nanoparticles and increasing the risk of side effects. In addition, some evidences of anti-PEG

immunological response have been reported.(15) To overcome the immune system barriers, as

34

well as to enhance the accumulation of nanoparticles in the tumor, new strategies to

functionalize nanomaterials have been developed. Among them, coating with natural cell

membranes has been shown to be an interesting alternative to camouflage the particles and

extend their blood circulation time. Red blood cell membrane (RBC)-coated polymeric

nanoparticles have been shown to present longer blood circulation time compared to the PEG-

coated ones.(16) Also, it has been demonstrated that CD47 proteins, a biomolecule capable of

inhibiting the phagocytosis, are transferred to the nanoparticle surface and get exposed,

allowing the nanomaterials to establish molecular interactions and reducing their

susceptibility to be captured by macrophages.(17) Using a cancer cell membrane, it may also

be possible to transfer multiple membrane-bound tumor-associated antigens to the

nanoparticles surface,(18) which can be particularly useful for applications in cancer cell

targeting (18) and immunotherapy.(19) Furthermore, the cell membrane coating may also be

explored in drug delivery systems,(20) allowing the fabrication of multifunctional platforms.

To address the challenge of precise location of theranostic nanoparticles in the body,

the development of near-infrared resonant, magnetic resonance imaging (MRI) active

theranostic nanocomplexes that enhance visualization by mainstream bioimaging techniques

is highly desired. MRI is currently the most universally used biomedical imaging

modality.(21) It is a non-invasive technique with contrast versatility, high spatial and

temporal resolution, that it not used harmful high-energy radiation (22) There are two main

types of MRI contrast agents currently in widespread clinical use. T2-weighted contrast agents

locally modify the spin-spin relaxation process of water protons, producing negative or dark

images (based on materials such as superparamagnetic Fe3O4 nanoparticles). T1-weighted

contrast agents affect nearby protons through spin-lattice relaxation, producing positive or

bright image contrast (based on paramagnetic materials such as Gd(III) and Mn(II)).(23)

Despite their utility, T2 contrast agents have several disadvantages that limit their

utility in clinical applications. They can cause reduction in the MRI signal, which can be

confused with other pathogenic conditions, such as blood clots and endogenous iron. In the

case of tumor imaging, they can induce magnetic field perturbations on the protons in

neighbor normal tissues, which can make spatially well-resolved diagnosis difficult.(24) In

contrast, T1 contrast agents increase the specificity and sensitivity of MR image. Among the

paramagnetic materials useful for T1 contrast MR imaging, Gd(III) is the most effective

contrast agent currently available for clinical use. However, Gd(III) ions exhibit high toxicity

in their uncomplexed state and Gd(III)-chelates such as DOTA and DTPA suffer from poor

sensitivity, stability, and rapid renal clearance.(22, 25-27) Considerable efforts have been

35

devoted to the incorporation of Gd(III) onto or into nanoparticles that will enhance their

sensitivity by increasing the concentration of Gd(III) per nanocarrier, prolonging imaging

time, and reducing their toxicity.(22, 28-32)

Based on the exposed, this thesis explores three main subjects:

In Chapter 2, we present a theoretical introduction and describe the synthesis and

characterization of three types of gold based-plasmonic nanoparticles: gold nanorods, gold

nanomatryoskas, and gold-coated gold sulfide nanoshells. We explored the dependence of the

surface plasmon resonance on parameters such as shape, size and composition. Gold nanorods

were synthesized using the seed-mediated method in presence of cetyltrimethylammonium

bromide (CTAB). Gold nanomatryoshkas, consisting in a gold core, an interstitial nanoscale

silica (SiO2) layer, and a thin gold shell (Au/SiO2/Au) were synthesized with high control of

the thickness of the interstitial silica layer and gold shell. For the synthesis of Au-coated Au2S

nanoshells two methods were investigated: one and two step processes. All particles were

characterized by electronic microscopy and spectroscopic techniques. The three systems

presented strong absorption in the near infrared region and maximum diameter of 100 nm,

two desirable characteristics for medical applications. Moreover, their gold surface allows

straightforward conjugation of polymers and biomolecules to the nanoparticle surface, which

is important to maintain the particle stability and add new functionalities for applications in

medicine.

In Chapter 3, we developed a multifunctional nanocarrier for photothermal therapy

and drug delivery. In this new nanotheranostic system, PEG-coated gold nanorods were

grouped into natural cell membrane vesicles from lung cancer cell (A549) membranes and

loaded with β-Lapachone (CM-β-lap-PEG-AuNRs). Our goal was to develop specific

multifunctional systems for cancer treatment by using the antigens and the unique properties

of the cell membrane combined with photothermal properties of AuNRs and anticancer

activity of β-lap. We have chosen A549 cell line, because it is a metastatic cancer type and,

consequently, may be good candidates to enhance delivery to the tumor and metastatic sites.

The platform was characterized by spectroscopic and scanning electron microscopy

techniques and its photothermal properties both in suspension and in vitro were investigated

using a NIR laser. Further, cytotoxicity experiments were performed to evaluate its safety in

the absence of laser irradiation.

36

In Chapter 4, we report a modification of gold nanomatryoskas that transforms them

into high-relaxivity MRI-active contrast agents as well as highly efficient NIR photothermal

transducers for photothermal cancer therapy. This was accomplished by incorporating Gd(III)

into the interstitial silica layer of the NM structure. This strategy adds MRI contrast

enhancement to the properties of these nanoparticles so they can be used in both bioimaging

diagnosis and therapeutic applications. Surprisingly, the sequestration of Gd(III) within the

inner layer of the nanoparticle resulted in increased T1 relaxivities relative to Gd(III)-based

chelates currently in widespread use. The enhanced MRI sensitivity is accompanied by

reduced toxicity, since the Gd(III) remains sequestered within the nanoparticle. This

combination of MRI active contrast within a photothermal therapeutic transducer with

superior properties should facilitate MR-based “see and treat” approaches that will enable

increased efficiencies and optimization of photothermal therapy treatment protocols of solid

tumors. This project was developed at Rice University (advisor: Dr. Naomi Halas) during the

International Research Internship (BEPE-DR FAPESP).

37

2 SYNTHESIS AND CHARACTERIZATION OF PLASMONIC

NANOPARTICLES

2.1 INTRODUCTION

2.1.1 Plasmonic nanoparticles

Plasmonic nanoparticles differ from other nanoplatforms such as semiconductor

quantum dots, magnetic and polymeric nanostructures by their unique surface plasmon

resonance (SPR). When a metal nanoparticle is exposed to the oscillating electromagnetic

field of the light, its conduction band electrons undergo a collective coherent oscillation in

resonance with the frequency of light.(33) This results in a strongly enhanced absorption and

scattering orders of magnitude higher than the absorption and emission of dye molecules.(33-

34) The coherent oscillation of the metal free electrons in resonance with the electromagnetic

field is the SPR.

The SPR band intensity and wavelength are theoretically described by the Mie theory

and depend on several factors affecting the electron charge density on nanoparticle surface

such as particle size, shape, metal type, structure, composition, and dielectric constant of the

surrounding medium.(35) Consequently, the resonant photon wavelength is different for

different metals, sizes and morphologies. A free Mie theory simulator is available at

http://www.nanocomposix.com/support/tools, and allows the prediction of the extinction

spectra for several types of plasmonic nanostructures, including core-shell systems.

The versatility and possibility to design systems with the desired optical properties by

manipulating the composition, shape and size allow the use of plasmonic nanostructures for a

wide range of applications. For example, plasmonic nanostructures of noble metals, mainly

silver and gold, have shown significant promise in the field of photocatalysis for direct

conversion of solar to chemical energy.(36-37) They present higher photocatalytic activity

compared to other metal structures when exposed to resonant photons of Sun-like intensities.

Some examples of the direct photocatalysis of plasmonic nanostructures include exothermic

partial oxidation, selective reduction, and organic decomposition reactions on excited

plasmonic Ag and Au nanostructures.(36) Plasmonic nanoparticles have also been

successfully incorporated into solar cell devices, where they enhance sunlight absorption and,

consequently, the conversion of solar energy into electricity. For example, it has been

38

demonstrated an increased optical absorption and photocurrent in thin film silicon solar cells

by the incorporation of silver and gold nanoparticles.(38-39)

Due to the combination of multiple scattering and optical absorption of light,

plasmonic nanostructures can heat a reduced fluid volume and may be used for distillation

applications.(40) Broadband light-absorbing plasmonic nanoparticles have also been used as

solar photothermal heaters for efficient solar autoclave, which could be useful for sanitation

of instruments and materials in resource-limited, remote locations.(41) The electron

oscillation around the plasmonic particle surface can also modify the emission properties of

fluorophores in their proximity, resulting in fluorescence enhancement or quenching.(42-43)

The biomedical field has been greatly benefited with the advances in

nanotechonology. Plasmonic nanoparticles have been used as contrast agents for optical

imaging because of their efficient light-scattering properties. Gold-based nanoparticles, for

example, have been demonstrated to provide high-contrast images of cancer cells using a

variety of optical techniques, including dark-field microscopy,(44) reflectance confocal

microscopy,(45) one- or two-photon fluorescence imaging,(46) surface enhanced Raman

scattering (SERS),(47-48) and photoacoustic imaging.(49) They also have shown interesting

properties for use in sensors for detecting different types of targets, such as proteins,(50)

specific DNA sequences (51) or cancer cells.(52)

The use of plasmonic nanoparticles as therapeutic agents has also opened new

opportunities for alternative treatments. The use of light as an external stimulus in the

presence of plasmonic nanoparticles has enabled the conversion of light into heat and

applications in efficient treatments, such as plasmonic photothermal therapy (PPTT) (53-54)

and light controlled drug release.(55) Besides the advantages of the high stability, the use of

plasmonic nanoparticles as photothermal agents has been proved to enhance the efficiency of

near infrared (NIR) light absorption, where maximum radiation penetration through tissue

occurs,(8) by several orders of magnitude compared to conventional phototherapy agents.(56)

Nanomaterials can take advantage of the enhanced permeability and retention effect (EPR),

presenting a higher accumulation in tumor sites compared to small molecules agents.(57) The

current trends go beyond the use of plasmonic systems as agents for simultaneous diagnostic

and therapy, the so-called theranostic systems.

39

2.1.2 Gold nanorods

Gold-based nanomaterials have become an important alternative for medical

applications due to their unique plasmonic properties combined with their potential non-

cytotoxicity and chemical/thermal stability.(58) The possibility of obtaining gold

nanoparticles in a wide variety of sizes, shapes and functionalizations allows the control of

their properties and open a broad range of possibilities for applications in detection and

treatment of diseases.(35, 58-59)

Special attention has been focused on rod-shaped gold nanomaterials, called gold

nanorods (AuNRs). As predicted by Gan theory in 1915, when the shape of Au nanoparticles

change from spheres to rods, the SPR band is split into two bands related to the coherent

motion of the conduction band electrons along the long and short axes of the particle.(35, 59-

60) The transversal band occurs in the visible region, while the longitudinal band usually

occurs in the near infrared region (NIR), which has maximum penetration through tissue,(8)

as shown in Figure 2.1.

Figure 2.1 - Schematic representation of the SPR oscillation and its respective extinction spectra for gold (A)

nanospheres and (B) nanorods.

Source: By the author.

Upon illumination, plasmonic nanoparticles will preferentially scatter or absorb light

or a combination of both. In medical applications, scattering is useful for contrast

B

A

400 500 600 700 800 900

Extin

ction

/ a

.u.

/ nm

400 500 600 700 800

Extinction / u

.a.

/ nm

e- e- e-e-

e-e- e-

e-

e- e-e-e-

e- e-e-e-

e- e-

e- e-

+ + ++

+ + + +

++ + +

++ + +

++

++

40

enhancement in bioimaging, while absorption is desired for photothermal therapy.

Quantitative description of the cross-sections of absorption (Cabs), scattering (Csca), and total

extinction (Cext) for gold nanomaterials are shown in Equations 2.1, 2.2. and 2.3 according to

Gans theory, or Mie-Gans theory, an extension of Mie theory for including spheroidal

particles.(33)

(2.1)

⁄ ∑

( ) ⁄

(

[ ( )

⁄

]

)

(2.2)

∑

( ) ⁄

(

[ ( )

⁄

]

)

(2.3)

Where λ is the wavelength of light, V is the unit of volume of the nanoparticle, m is the

dielectric constant of the surrounding medium, and is the dielectric constant of the metal

given by = 1 + i2 (1 and 2 are the real and imaginary components of the dielectric

constant, respectively).(33) Finally, ni is the depolarization factor defined by:(33)

(

√

√

√ ) (2.4)

(2.5)

Where a, b, and c are the theree axes of the particle, a>b=c, and R is the aspect ratio a/b. For

spheres, ni = 1/3, resulting in only one SPR band. The SPR occurs when ( )

⁄ where i = a for the logitudinal and i = b,c for the transverse resonance.(33)

The equations show that the SPR depends not only on the wavelength of the light, but

also on the dielectric constant of the surrounding medium as well as some characteristics of

the particle such as dielectric constant, size and aspect ratio. Link et al found a linear

41

relationship between the longitudinal SPR absorption maximum and the aspect ratio of

nanorods:(61-62)

(2.6)

In aqueous solution , the equation can be written as following:

(2.7)

The Cext (in cm2) can be related to Beer-Lambert’s law and can be used to calculated

the concentration according to Equation 2.8. For gold nanorods with aspect ratio around 3 (a

~ 49 and b ~ 16 nm) the extinction cross section (Cext) of the longitudinal band is around

5x10-12

cm2, while for an aspect ratio of about 3.33 (a ~ 50 nm and b ~ 15 nm) is 8.4x10

-12

cm2.(63)

(2.8)

Where N is the concentration of particles (number of particles/mL), A is the experimental

extinction in arbitrary units, and l is the optical path of the cuvette in cm. The Cext (in cm2)

can also be related to the coeficient of extintion from Beer-Lambert’s law:

(2.9)

2.1.3 Gold nanoshells and nanomatryoshkas

Recent advances in the chemical synthesis of nanomaterials have led to the fabrication

of multilayered nanostructures, a unique and important class of nanomaterials of intense

technological interest due their ability to manipulate light. In particular, gold nanoshells,

consisting of a silica nanoparticle core surrounded by a thin gold shell layer (SiO2/Au), have

offered exciting potential for applications in a broad range of spectroscopic, photonic, and

biomedical applications.(42,47-48,64-66) A schematic of the gold nanoshell and a

42

representative UV-VIS-NIR spectrum are shown on Figure 2.2. Their surface plasmon

resonance can be tuned to the near-infrared region by changing the thickness of the gold layer.

The shoulder around 600 nm is an indicative of the complete gold shell layer formation.(67)

Figure 2.2 - Schematic representation of a gold nanoshell and a representative extinction spectrum for a [r1, r2] =

[62, 76] nm.

Source: By the author.

For simple gold nanostructures, the absorption and scattering efficiencies depend

mainly on nanoparticles size and shape, as described previously. For layered systems, this

relationship becomes more complex and geometry dependent.(67) The tunable optical

properties of gold nanoshells have been explained by the plasmon hybridization model.(68)

This theory deconstructs a complex structure into two elementary structures and analyses the

interactions between the individual plasmons in the hybridized structure.(68)

As discussed earlier, Mie theory predicts the extinction spectra of plasmonic systems,

and, consequently, their plasmon resonance frequency. For a gold sphere and sphere cavity,

the plasmon resonance frequencies predicted from Mie theory can be described as:

(

)

⁄

(2.10)

(

)

⁄

(2.11)

Where ωs,l and ωc,l are the plasmon frequencies of the gold sphere and cavity, respectively; ωB

is the plasmon frequency of the bulk metal, and l is the angular moment. The oscillation of

r1

r2

400 600 800 1000

Extin

ction

(a.u

.)

Wavelength (nm)

43

the conduction band electrons in the inner and outer surfaces of the gold shell results in the

hybridization of the cavity and sphere plasmons (Figure 2.3).

Figure 2.3 - Schematic energy-level diagram describing the plasmon hybridization in gold nanoshells resulting

from the interaction between the sphere and cavity plasmons

Source: PRODAN et al.(68)

Prodan et al showed that the interaction of the plasmons on the inner and outer

surfaces of the shell gives rise to two hybridized plasmon modes |ω+〉 and |ω–〉 for each l >

0, which corresponds to the antisymmetric and symmetric coupling between the two modes,

and can be described as:(68)

{

[ (

)

]

⁄

} (2.12)

From the equation, it is clear that the coupling depends on the ratio r1/r2, which

corresponds to the radius of the silica core and entire particle (Figure 2.2), and explains the

tunable optical properties of gold nanoshells. Consequently, the strength of the coupling

between the cavity and sphere plasmon depends on the thickness of the gold shell, i.e.,

decreasing of the gold shell thickness results in a shift to lower energy of the plasmon

resonance.(68) This plasmon hybridization theory can also be extended to understand the

plasmon response of more complex metallic nanostructures, such as dimers or nanoparticle

44

assemblies. As shown previously for gold nanorods, the extinction cross-sections and the

respective absorption and scattering cross section of gold nanoshells can also be derived from

Mie theory, and be used for determination of particle concentration (Equation 2.8). In this

case, we have to consider that the particle consist of three regions with distinct dielectric

properties: the silica core, the gold shell, and the surrounding medium.(69)

Although many successful applications have been achieved with gold nanoshells,(7)

their 150 nm diameter range necessary to have a good NIR activity may limit some of their

biomedical applications.(10) Au-coated Au2S nanoshells present a configuration similar to

Au-coated SiO2 nanoshells, i.e., a dielectric core coated with a metal shell, but usually with a

diameter around 50 nm. Analogously to gold nanoshells, this system also present a shift in the

plasmon resonance band to NIR, and therefore are of technological interest for many

applications.(70)

Recently, multilayered gold nanoparticles, consisting of a gold core, an interstitial

nanoscale SiO2 layer, and a thin gold shell layer (Au/SiO2/Au) have offered the possibility to

achieve NIR plasmon resonances in the sub-100 nm size range (Figure 2.4).(10,53) They

were called nanomatyoshka in analogy to matryoshka dolls, and similarly to gold nanoshells,

the hydridization theory has been used to explain the optical properties of gold

nanomatryoshkas. According to Bardhan et al, the strong coupling between the plasmons

supported by the gold core and the gold shell allows the tuning of the plasmon resonance to

the NIR region in particles with smaller dimensions than the gold nanoshell.(67)

Figure 2.4 - Schematic representation of a gold nanomatryoshka and a representative extinction spectrum for a

[r1, r2, r3] = [24, 34, 44] nm.

Source: By the author.

The optical properties of gold nanomatryoshkas (NM) can be tuned by modifying the

relative radius (r1, r2, and r3) in the particle.(67) For example, maintaining the particle size, an

r1 r2

r3

400 600 800 1000

Extin

ctio

n (

a.u

.)

Wavelength (nm)

45

increase in the gold core diameter and a decrease in the silica layer thickness increase the

coupling between the core and shell, resulting in higher absorption efficiency.(67) These

properties make them extremely versatile and promising for a broad range of optical and

biomedical applications. Besides the biocompatibility, and facile surface conjugation

chemistry,(71) this system has shown several advantages compared to others NIR

photothermal transducers. Ayala-Orozco et al. demonstrated a 4–5 fold increase in

nanomatryoshkas uptake by a triple negative breast tumor model, and consequently

nanomatryoshkas presented an improved photothermal therapy efficacy.(53) They attributed

this enhancement to a more suitable diameter of nanomatryoshkas (~100 nm) when compared

to nanoshells (~150 nm).

2.2 EXPERIMENTAL SECTION

2.2.1 Materials

All reagents were of analytical grade and were used without further purification. The

solutions were prepared using ultrapure water (18.2 MΩ cm) and the glassware were cleaned

with aqua regia and thoroughly washed with distilled and Milli-Q water.

Cetyltrimethylammonium bromide (CTAB), silver nitrate (AgNO3), tetrachloroauric acid

(HAuCl4), sodium borohydrate (NaBH4) and ascorbic acid were purchased from Sigma-

Aldrich. Tetraethoxysilane (TEOS) and 3-aminopropyl)triethoxysilane (APTES) were

purchased from Gelest. Tetrakis(hydoxymethyl) phosphonium chloride (THPC), chloroauric

acid (HAuCl4.3H2O, and Na2S were purchased from Sigma Aldrich; anhydrous potassium

carbonate (K2CO3) from Fisher, NaOH from Fisher. 50 nm gold colloid citrate NanoXact was

purchased from NanoComposix and 200 proof ethanol was purchased from KOPTEC.

2.2.2 Gold nanorods (AuNR) synthesis

Gold nanorods (AuNRs) were synthesized by the seed-mediated method in the

presence of the surfactant CTAB.(72) The seeds were prepared by adding 250 µL of

HAuCl4.3H2O 0.01 mol L-1

in 7.5 mL of CTAB aqueous solution 0.1 mol L-1

under gentle

stirring. Next, 600 µL of iced cold NaBH4 0.1 mol L-1

was added and the system was kept

under vigorous stirring for 10 min. The color of the solution changed from yellow to pale

46

brown. The solution was kept at 25ºC for about 2 hours before using. The final concentrations

of the reagents in the final growth solution were adjusted to obtain gold nanorods with

different aspect ratios. Briefly, between 3 - 4 mL of 0.01 mol L-1

HAuCl4.3H2O were mixed

with 47.5 mL of 0.1 mol L-1

CTAB aqueous solution, followed by addition of different

amount (300-600 µL) of 0.01 mol L-1

AgNO3 and 480 µL of 0.01 mol L-1

ascorbic acid

solutions. The color of the system changed from dark yellow to transparent, indicating the

reduction of Au3+

to Au+. Finally, different amounts of the seed solution (between 70-100 µL)

previously prepared were added and the system was kept under gentle stirring for 20 min. The

system was left at room temperature and protected from light for 4 h, and the supernatant was

washed by centrifugation twice at 9000 g for 5 min (Spinlab SL-5GR).

2.2.3 Gold nanomatryoskas (NM) synthesis

Gold nanomatryoshkas (NM) were synthesized by improving a previously Halas

group’s reported method.(10) Briefly, 21 mL of 0.05 mg/mL 50 nm gold colloid were mixed

with 180 mL of 200 proof ethanol, 1.8 mL of 28-30% ammonium hydroxide, 36 µL 10%

TEOS solution in ethanol, and 36 µL of 10% APTES in ethanol solution. The reaction was

allowed to proceed for 65 min at room temperature under vigorous stirring and then stored in

the refrigerator at 4°C for 22 hours. After that, the Au/SiO2 nanoparticle suspension was

dialyzed in 200 proof ethanol for about 16 hours at room temperature. The nanoparticle

suspension was cooled to 4ºC, centrifuged at 1500 rcf for 35 min and re-dispersed by

sonication in a total volume of about 3 mL of ethanol. The Au/SiO2/Au nanoparticles were

prepared by decorating the silica coated nanoparticles with small gold colloid (2-3 nm)

fabricated by the method reported by Duff et al.(73) Briefly, Duff colloids were prepared by

mixing 2.4 mL of 1 mol L-1

NaOH and 8 mL of 1.2 mmol L-1

aqueous THPC solution in 360

mL of Milli-Q water. After stirring for 5 min, 13.5 mL of 1% (m/v) HAuCl4 were added and

the solution immediately turned brown. The final Duff colloids were refrigerated for 1 week

before use. Next, 1 mL of Au/SiO2 nanoparticles was mixed with 600 µL of 1 M NaCl and

40 mL of Duff gold colloid (1-2 nm). The precursor particles were left unperturbed for 24 h

at room temperature, followed by sonication and centrifugation (950 g, 30 min) to remove the

excess of Duff gold colloid. The pellet was dispersed in 2 mL of Milli-Q water and

centrifuged two more times at 800 g for 20 minutes. The final precipitate was dispersed in 500

µL Milli-Q H2O and it is called the seeded precursor. The formation of a continuous metallic

47

shell around the seeded precursor was performed by mixing 3 mL of plating solution (1%

HAuCl4- K2CO3 solution previously prepared) with 20-40 µL of the seeded precursor and 15

µL of formaldehyde, under a fast shaking for 1 minute. The color of the solution changed

from reddish to purple upon the formation of the gold outer shell.

2.2.4 Au-coated Au2S nanoshell synthesis

One- step synthesis: The synthesis was performed by mixing HAuCl4 and Na2S according to

an improvement of a previously reported method.(70, 74) Different ratios of HAuCl4 and

Na2S were tested and the one that gave the best NIR absorption was chosen for the following

experiments. In this case, 10 mL of HAuCl4 2 mmol l-1

were mixed with 18 mL of Na2S 1

mmol L-1

, and the system was kept protected from light for 12 hours. The particles were then

centrifuged at 200 g for 30 minutes in a 50 mL centrifuge tube containing 14 mL, and

resuspended in Milli-Q water.

The separation of the Au-coated Au2S nanoshells from the plates was carried out in a

density gradient according to a previously reported method.(75) For this, 10, 20, 30, 40, 50,

and 60 % w/v aqueous solutions of OptiPrep Density Gradient Medium (Sigma-Aldrich) were

prepared and carefully placed in a 50 mL centrifuge tube from the most to the least dense (5

mL of each one). The nanoparticles were then placed at the top of the gradient and the system

was centrifuged at 22000 g at 23ºC for 15 min using a Beckman Coulter Optima L-90k

Ultracentrifuge with a SW32 rotor (Swinging Bucket). Finally, the fractions were collected,

and each individual fraction was purified by centrifugation and resuspended in Milli-Q water.

The fractions were characterized by UV-VIS-NIR spectroscopy and Transmission Electron

microscopy (TEM).

Two-step synthesis: First, small gold sulfide (Au2S) nanoparticles were synthesized by

modification of a previously reported method.(76) For this, 5 mL of HAuCl4 2 mmol L-1

were

mixtured with 100 µL of NaOH 3 mol L-1

solution. The solution became immediately

colorless indicating the reduction of Au3+

to Au+. Next, 6 mL of Na2S 1 m mol L

-1 were added

and the solution became yellowish-brown after one hour, demonstrating the formation of the

Au2S nanoparticles. The second step consisted in the fabrication of the gold shell around the

Au2S particles. We used a procedure similar to that used for fabrication of the Au shell of the

gold nanomatryoshkas was employed. In this case, the Au2S nanoparticles were used as

48

seeded precursor and the formation of a continuous metallic shell was performed by mixing 3

mL of plating solution (1% HAuCl4- K2CO3 solution previously prepared) with 100-250 µL

of Au2S nanoparticles, followed by 15 µL of formaldehyde under a fast shaking for 1 min.

The color of the solution changed from yellowish-brown to purplish or greenish upon the

formation of the gold outer shell and depended on the volume used.

2.2.5 Characterization Techniques

Ultraviolet–visible–near infrared (UV–VIS–NIR) spectroscopy was used to evaluate the

optical properties of the nanomaterials. The measurements were carried out on Hitachi U-

2900 spectrometer using a 1 cm quartz cell.

Transmission Electron Microscopy (TEM) is an important technique for directly imaging

nanomaterials, allowing quantitative measurements of size, distribution and morphology. The

images were taken in a JEOL JEM-2100 at 200 kV (LNano, CNPEM, Campinas-SP) or a

JEOL 1230 operating at 80 kV (Rice University). For this, one drop of each diluted sample

was deposited on a carbon film copper grid 300 mesh (Ted Pella, Inc.) and dried at room

temperature. High Resolution TEM coupled with energy dispersive spectroscopy (EDS) was

used to obtain the local elemental composition of the Au-coated Au2S nanoshells.

2.3 RESULTS AND DISCUSSION

2.3.1 Gold nanorod synthesis and characterization

Gold nanorods (AuNRs) were synthesized by the seed-mediated method in presence of

the surfactant CTAB,(72) and an overview of the procedure is illustrated in Figure 2.5. Small

gold nanoparticles (seeds) were initially prepared by rapid reduction of Au3+

in presence of

CTAB (Figure 2.5A). The characterization of the seeds is shown in Figure 2.6. From the

UV-VIS-NIR spectrum, it is possible to observe a low-intensity band at 515 nm, typical of

very small gold nanoparticles.(77) The diameter measured by dynamic light scattering (DLS)

and transmission electron microscopy (TEM) image reveals particles with an average

diameter of about 2 nm (Figure 2.6B-C).

49

Figure 2.5 - Schematic representation of the gold nanorod synthesis process. (A) First, small gold nanoparticles

are synthetized in the presence of CTAB, and (B) used as seeds for the growth of the nanorods.

Source: By the author.

Figure 2.6 - Characterization of the seeds: (A) UV-VIS-NIR spectrum, (B) Hydrodynamic diameter obtained

from Dynamic Light Scattering measurements, and (C) TEM image, showing very small particles.

Source: By the author.

Next, the small gold particles were used as seeds for the anisotropic growth (Figure

2.5B). Figure 2.7A shows the growth kinetics of nanorods monitored by UV-VIS-NIR

spectroscopy. The spectrum is typical of AuNRs, which exhibit two surface plasmon

resonances (SPR): one around 500 nm due the coherent motion of the conduction band

electrons along short axes (transversal band), and the other at higher wavelengths

corresponding to the oscillation along long axes of the particle (longitudinal band).(35, 59,

60) As it can be observed, the longitudinal plasmon band appears in the first minutes and its

intensity increases with the time and becomes blue shifted. This shift of the longitudinal band

is related to the modification of the aspect ratio during the growth process,(72) and after about

40-50 min, the reaction is almost complete (Figure 2.7B).

NaBH4N+

Br -

CH3

CH3

CH3

HAuCl4

HAuCl4Ascorbic Acid

AgNO3

N+

Br -

CH3

CH3

CH3

A

B

0 1 2 3 4 5 6 70

5

10

15

20

25

Inte

nsity %

Hydrodynamic diameter / nm400 500 600 700 800

Abso

rba

nce

/ a

.u.

/ nm

10 nm

A B C

50

Figure 2.7 - (A) UV-VIS-NIR spectra showing the growth kinetics of nanorods after addition of 100 µL of seeds

into the growth solution, and (B) the changing in the maximal wavelength of the longitudinal band

as function of time.

Source: By the author.

Figure 2.8 shows the UV-VIS-NIR spectra and their correspondent TEM images of

AuNRs obtained using different amount of AgNO3. In all cases, we used 100 µL of seed

suspension. It is clear that the silver salt plays a significant role in the growth of the nanorods.

Higher silver nitrate concentration yields higher aspect ratios (length/width). It is interesting

to note that a small variation on the gold nanorods size is enough to cause a significant shift in

the longitudinal plasmon band. Figure 2.9 displays low-magnification TEM images of

AuNRs with maximal longitudinal wavelength at 700 nm, revealing a very homogeneous

system.

400 500 600 700 800 9000.0

0.5

1.0

1.5

Extin

ction

/ a

.u.

/ nm

0 10 20 30 40 50 60

680

720

760

800

m

ax lo

ng

itu

din

al / n

m

Time / min

A B

51

Figure 2.8 - UV-VIS-NIR spectra of AuNRs obtained using different amount of AgNOs and the respective TEM

images. The aspect ratio are 2.1, 2.8, and 3.5 for blue, green, and red, respectively. Scale bar = 50

nm.

Source: By the author.

Figure 2.9 - TEM images of the gold nanorods with maximal longitudinal absorption at 700 nm, showing a

monodisperse system.

Source: By the author.

We have observed in our experiments and other authors have also reported that the use

of AgNO3 produces not only higher aspect ratio nanorods, but also increases the yield of the

reaction.(35) In other words, when AgNO3 is not used in the seed-mediated method, the

resulting suspension presents a large amount of spherical gold particles. The mechanism of

Ag+

ions in the nanorods synthesis has been widely investigated in the last years. It is known

400 500 600 700 800 900E

xtin

ction

/ a

.u.

/ nm

AgNO3

50 nm

A B

52

that the silver ions are not reduced in the synthesis conditions, since the ascorbic acid could

only reduce the silver in a high pH, which is not the case.(35, 78) Sal et al. proposed that

AgNO3 forms AgBr in the presence of CTAB, which in turn adsorbs differentially in the faces

of gold seeds, restricting the growth in one direction.(72) Another study suggest that Ag+

ions

decrease the rate of growth and increase the energy in the sides (110) of monocrystalline gold

nanorods, i.e., the ions adsorb preferentially in the sides with specific structures and direct the

anisotropic growth.(79) Nikoobakht et al proposed that AgBr decreases the charge density

and, consequently, the repulsion between the polar headgroups of CTAB resulting in a

template for the growth in that direction.(78)

Recently, a detailed investigation of the mechanism of the anisotropic growth of the

gold nanorods by direct observations in the atomic scale using electronic microscopy has been

reported.(80) According to the paper, the seeds prepared in presence of CTAB have a

cuboctahedral morphology, bound by symmetrically arranged (111) and (100) surfaces.

Initially, when these seeds are introduced in the growth solution, they grow isotropically until

4-6 nm in diameter. Then, small truncating surfaces consisting of a few atoms start to form

nonuniformly at the intersection of (111) facets. These truncations are a more open atomic

structure than the comparatively close-packed (111) and (100) surfaces already present in the

nanoparticle, and consequently are potentially preferred sites for Ag+ underpotential

deposition, preventing further Au deposition and resulting in the onedirectional growth.

The aspect ratio may also be controlled by changing the amount of seeds in the growth

media and keeping the AgNO3 amount constant (Figure 2.10). As the number of seeds in the

media increases, the longitudinal wavelength increases to higher wavelength. However,

particles with absorption at higher wavelengths could not be fabricated, which highlights the

importance of silver nitrate, especially for high aspect ratio AuNRs.

Figure 2.10 - UV-VIS-NIR spectra of gold nanorods prepared with different concentrations of seeds.

Source: By the author.

400 500 600 700 800 900

Extin

ction

/ a

.u.

nm

Seeds

53

2.3.2 Gold nanomatryoshkas synthesis and characterization

Gold nanomatryoshkas (NM) were fabricated as shown in Figure 2.11. Gold

nanoparticles of radius 50 ± 4 nm were initially coated with a ~ 21 nm amorphous silica layer

doped with (3-aminopropyl)triethoxysilane (APTES). The SiO2 layer were growth based on

the Stöber method, a condensation reaction of tetraethyl orthosilicate (TEOS) catalyzed by

ammonium hydroxide in ethanol:water mixture.(81) The function of the APTES is to

introduce amine groups, which in turn will allow the attaching of small gold colloids (2-3 nm

diameter) onto the surface of the silica layer in the next step. The small gold colloids were

prepared in a separate reaction using tetrakis (hydroxymethyl)phosphonium (THPC) as a

reducing agent following the method reported by Duff et. al.(73) To make the complete shell,

the SiO2-coated gold nanoparticles decorated with Duff colloids, called seeded precursor,

were added to the right volume ratio of Au3+

aqueous solution containing potassium carbonate

(plating solution). Finally, formaldehyde is added under vigorous stirring to reduce the Au3+

to metallic gold which grows onto the Duff colloids to form a complete Au layer, ie, the Duff

colloids serve as nucleation sites for the formation of Au shell layer.

Figure 2.11 - (A) Schematic representation of the gold nanomatryoshka synthesis: 50 nm diameter gold colloids

are coated with SiO2-APTES shell, followed by a continuous gold shell.

Source: By the author.

The extinction spectra and their respective TEM images corresponding to three stages

of the synthesis are shown on Figure 2.12. We can see that the resulting nanomatryskas

present average dimensions of [r1, r2, r3] = [25, 38, 53] nm and plasmon resonance band

around 800 nm, which is highly desired for photothermal therapy applications because of the

maximum penetration depth of light in biological tissue.(8) The critical step in the synthesis

was to obtain the right silica layer thickness to guarantee a controlled hybridized plasmon

resonance and consequently the absorption at the desired wavelength. Nanomatryoshkas with

different gold shell thicknesses can be fabricated by varying the volume ratio of the seeded

precursors and the plating solution. By increasing the seeded precursor from 30 to 34 µL, it

resulted in a thinner gold shell and red-shifting of the plasmon resonance (Figure 2.13).

2 nm AuNPTEOS, APTES

CH2O

Au colloid SiO2-coated Au colloid Seeded precursor NM

Au3+

54

However, when the volume of seeded precursor was increased to 40 µL, plasmon resonance

band did not change anymore, instead the peak around 600 nm increases, indicating that the

amount of seeds was too high, and some NM with incomplete Au shell layer starts to form.

400 500 600 700 800 900 1000

E

xtin

ction

/ a

.u.

/ nm

400 500 600 700 800 900 1000

Extin

ctio

n / a

.u.

/ nm

B

A

C

400 500 600 700 800 900 1000

Extin

ction

/ a

.u.

/ nm

Figure 2.12 - UV-VIS-NIR extinction spectra and their respective TEM images corresponding to (A) Au

colloids of about 50 nm, (B) APTES-SiO2-coated Au colloids, and (C) gold nanomatryoskas.

Source: By the author.

55

Figure 2.13 - UV-VIS-NIR spectra of gold nanomatryoskas obtained using different amount of seeded

precursor.

Source: By the author.

2.3.3 Au-coated Au2S nanoshells

Au-coated Au2S nanoshells were initially prepared according to a previous reported

one step method.(70, 74) Averitt et al proposed the following reaction for the reduction

process leading to the growth of Au-coated Au2S nanoshells:(74)

2AuCl4- + HS

- Au2S + H

+ +8Cl

-

2AuCl4- + 3HS

- 2Au + 3S + 3H

+ +8Cl

-

According to the reaction, Au and S will be available for subsequent nucleation and

growth, and since the synthesis is performed in one step, it is believed that both Au and Au-

coated Au2S nanoshells grow simultaneously. Figure 2.14 shows the UV-VIS-NIR spectrum

and the respective TEM image. The absorption peak at 520 nm is due to the plasmon

resonance of gold colloid, while the second peak has been attributed to the Au-coated Au2S

nanoshells.(70, 74) Au-coated Au2S nanoshells prepared by this method presented an average

diameter of 40 nm. However, the TEM images revealed the presence of not only Au-coated

Au2S nanoshells but also triangular plates. The local atomic composition of the nanoshells

and triangular plates were analyzed by High Resolution TEM coupled with energy dispersive

spectroscopy (EDS) (Figure 2.15). The Figure shows the TEM image and the respective

localization of the EDS analysis for Au-coated Au2S nanoshells and the triangular plates. The

column plots correspond to the percentage calculation from the EDS spectrum. The results

400 600 800 1000

Extinction / a

.u.

/ nm

30 L

34 L

40 L

56

show that the spheres are composed by both Au and S, in a proportion suitable for a Au-

coated Au2S, while the triangular plates are only constituted by gold.

Figure 2.14 - UV-VIS-NIR spectrum of Au-coated Au2S nanoshells and its respective TEM image.

Source: By the author.

Figure 2.15 - Analysis of the composition of the (A) nanoshells and (B) triangular plates by High Resolution

TEM coupled with energy dispersive spectroscopy (EDS).

Source: By the author.

The nanoshells were separated from the plates by a density gradient according to a

previously reported method.(75) Density gradient methods have been shown to be a simple

and effective way to separate nanomaterials with different size and shapes.(82) Figure 2.16

shows the pictures of the gradient before and after centrifugation and the TEM images and the

respective UV-VIS-NIR spectrum of the two indicated fractions. As it can be seen, the

triangular plates are more concentrated in the denser fraction, while the less dense one is

400 600 800 1000 1200

E

xtin

ctio

n /

a.u

.

/ nm

20 nm

A B

BA

57

constituted mostly by spherical particles. The UV-VIS-NIR also revealed significant

difference between the optical properties of both systems. Although a good separation had

been obtained, the difficult to remove the density gradient solution from the nanoparticles has

led to the aggregation of the system, limiting their biomedical applications.

Figure 2.16 - Pictures of the centrifuge tubes before and after the centrifugation showing the separation of the

particles through the density gradient, and the UV-VIS-NIR spectra and TEM images of the two

indicated fractions.

Source: By the author.

Different strategies to improve the synthesis and overcome the polydispersity problem

of the synthesis were tested and are summarized on Figure 2.17. The changing of Na2S to

Na2S2O3 results in a broader peak in the NIR, and the TEM image revealed the presence of

even bigger plates (Figure 2.17A). The use of an acid pH or N2 atmosphere during the

reaction improved the homogeneity of the sample; however the absorption at NIR decreased

significantly, and, in the case of the acid pH, the particles diameter were bigger than 200 nm

(Figures 2.17B-C). The addition of Na2S in two steps was also performed and, although we

observed a reduced number of triangular plates in the sample, the NIR band intensity was

lower (Figure 2.17D).

400 500 600 700 800E

xtin

ctio

n /

a.u

.

nm

400 500 600 700 800

Extinction / a

.u.

/ nm

Before centrifuge After centrifuge

100 nm

100 nm

58

Figure 2.17 - UV-VIS-NIR spectra and their respective TEM images of different synthesis process to

synthesized Au-coated Au2S nanoshells. (A) Na2S2O3:HAuCl4 (18 mL:10 mL); (B) Na2S :

HAuCl4 (18 mL : 10 mL) under N2 atmosphere; (C) Na2S : HAuCl4 (18 mL : 10 mL) in pH =

1.6, and (D) Na2S : HAuCl4 (10 mL : 10 mL), and after 5 min, more 2 mL of Na2S were added.

Source: By the author.

The synthesis in two steps seemed to be the best alternative for controlled fabrication

of Au-coated Au2S nanoparticles. In this way, a new strategy of was developed. Basically, the

synthesis was performed in two steps: Au2S nanoparticles were synthesized first, and then

coated with a gold shell. The Au2S NPs synthesis were performed by modification of a

previous reported method by Morris et al.(76) They have performed the reaction by mixing 10

mL of 2 mmol L-1

Na3Au(SO3)2 and 12 mL of fresh 1 mmol L-1

solution of Na2S.9H2O.(76)

Both solutions were coreless before reaction, and after 24 h, the color of the solution had

400 600 800 1000 1200

Extinction /

a.u

.

/ nm

400 600 800 1000 1200

Extinction / a

.u.

nm

400 600 800 1000 1200

Extinction / a

.u.

nm

50 nm

50 nm

400 600 800 1000 1200

Extinction /

a.u

.

/ nm

50 nm

50 nm

A

B

C

D

59

changed to yellowish-brown. The authors proposed that Au2S is formed by rapid nucleation to

form very small AunSm- clusters, followed by a very slowly growth. In our experiments,

instead of Na3Au(SO3)2, we have used HAuCl4. The main difference between these two

reagents is the oxidation state of the gold, while in Na3Au(SO3)2 it is Au+, in HAuCl4 is Au

3+.

To evaluate the influence of the oxidation state of the gold in the synthesis reaction, the gold

ions of the HAuCl4 were reduced from Au3+

to Au+

in presence of NaOH, the color of the

solution changed from yellow to colorless, indication the reduction.(83) Then, Na2S was

added to the solution and, after 1h30min the color of the solution changed from colorless to

yellowish-brown (inset of Figure 2.18). The Au2S NPs spectrum is similar to the one

obtained by Morries et al, as well as to other metal chalcogen nanoparticles such as Cu2S and

Ag2S, and it is typical for semiconductor nanoparticles with an indirect optical band gap

(Figure 2.18A). Therefore, it is clear that the product of the reaction between gold salt and

Na2S critically depends on the oxidation state of the gold salt. The TEM image revealed the

production of very small nanoparticles with size around 5 nm.

Figure 2.18 - UV-VIS-NIR spectrum and the respective TEM image taken 1h30 after mixing HAuCl4 with

NaOH and then with Na2S.

Source: By the author.

The resulting Au2S particles were then coated with a gold shell layer, according to

what was described in the experimental section. The UV-VIS-NIR shown in Figure 2.19 is

very similar to the one obtained for Au-coated SiO2 nanoshells (Figure 2.2), and the particles

presented an average diameter around 70 nm. The TEM image also revealed that some

particles present branches (inset of Figure 2.19). This is probably due to the use of NaOH in

the reaction, which has been proved to induce the formation of branched gold

nanoparticles.(83) Branched nanosystems have received considering attention in the last years

due to the interesting and distinctive optical and electronic properties that they present, such

50 nm

400 600 800 1000 1200

Extin

ctio

n /

a.u

.

/ nm

BA

60

as gold nanostars.(84) By changing the reduction agent of the reaction, we might be able to

produce non-branched systems.

Figure 2.19 - UV-VIS-NIR spectrum and the respective TEM image of the Au-coated Au2S nanoshells prepared

by a new two-step method.

Source: By the author.

2.4 CONCLUSIONS

We have presented the synthesis and characterization of three types of gold based-

plasmonic nanoparticles, and the dependence of the surface plasmon resonance with

parameters such as shape, size and composition. Gold nanorods were synthesized using the

seed-mediated method in presence of CTAB with high control over size and shape. The

resulting particles were monodisperse and the longitudinal plasmon resonance wavelength

was red-shifted by increasing the ratio length/width, called aspect ratio. Gold

nanomatryoshkas, consisting in a gold core, an interstitial nanoscale SiO2 layer, and a thin

gold shell layer (Au/SiO2/Au) were synthesized with high control over the thickness of the

interstitial silica layer and gold shell, and, consequently, their optical properties. The particles

were also monodiperse with a final diameter around 100 nm and a strong NIR absorption.

A promising Au-coated Au2S plasmonic material was also investigated. Our results

have shown that the one step synthesis usually used to fabricate these particles do not enable a

good control over size, distribution, and composition of the nanoshells. The resulting particles

were composed not only of Au-coated Au2S nanoshells but also of Au triangular plates

according to energy dispersive spectroscopy analysis-coupled with transmission electron

microscopy analysis. Density gradient was used to separate both systems, and although a good

separation had been obtained, the difficult to remove the density gradient solution from the

nanoparticles may limit their biomedical applications. Different methodologies were tested to

50 nm 50 nm

A B

400 600 800 1000 1200

Extin

ctio

n /

a.u

.

/ nm

61

improve the yield of the synthesis, and the two step method, in which Au2S small particles are

first synthesized and then covered with a gold layer, has provide promising results.

All systems presented strong NIR absorption and maximum diameter around 100 nm,

characteristics very relevant for medical applications. Moreover, their gold surface allows

straightforward conjugation of polymers and biomolecules to the nanoparticle surface, which

is important to maintain the particle stability and add new functionalities for applications in

medicine.

62

63

3 PLASMONIC GOLD NANORODS COATED WITH CANCER CELL

MEMBRANE AS A MULTIFUNCTIONAL SYSTEM FOR CANCER

THERAPY

3.1 INTRODUCTION

3.1.1 Theranostic nanoparticles

Cancer has been one of leading causes of death in the world. According to the

GLOBOCAN, project of the international agency for research on cancer, 14.1 million cases of

cancer were estimated in 2012 and this number is expected to increase to about 19.3 million in

ten years.(1) The current treatment for cancer include surgery, chemotherapy or radiation,

which led to many side effects.(85) In this scenario, the search for new drugs and methods to

enhance the early diagnosis and treatment of cancer is one of the major challenges in

medicine today.

The combination between the medicine and nanotechnology has been offered exciting

potential in the search for more efficient approaches against cancer. The golden standard in

this area is the possibility to fabricate multicomponent nanomaterials with different properties

that can be explored simultaneously in multiple applications.(86) In contrast to the traditional

approach that uses separate materials and tools for diagnosis and therapy, nanomedicines have

the potential to revolutionize the fight against cancer by integrating imaging agents and

therapeutic drugs in one nanometer scale complex.(87) These so-called “theranostic”, enable

detection and treatment in one single procedure and are emerging as an alternative to

independently administered traditional strategies.(87)

The field of theranostics has been rapidly increasing due to the versatility of

nanoparticles-based systems. Their unique optical, electronic and magnetic properties

combined with the possibility to incorporate multiple types of molecules onto a single

nanoparticle surface have enabled the design of many systems with different combined

functionalities. The combination of functions can also provide important information about

biodistribuition and mechanisms, opening opportunities to create more efficient

nanoplatforms.

64

3.1.2 Plasmonic Photothermal therapy (PPTT)

The use of laser light has offered exciting potential for applications in cancer therapy.

However, to achieve therapeutic efficacy, high laser power densities are required, leading to

the damage of both tumor and normal tissues.(34) To overcome this challenge, some agents

are employed to improve the laser efficacy and selectivity, allowing the use of reduced

irradiation energy. Transduction of light by specific agents into other types of energy such as

chemical and thermal energy has provided the basis for the development of efficient treatment

approaches such as photothermal therapy (PTT) and phodynamic therapy (PDT). In PTT,

when the agent absorbs the light, electrons make transitions from the ground to the excited

state. The relaxation through nonradiative process led to an increased kinetic energy and,

consequently, an increase on temperature of the local medium around the absorbing

species.(34,88)

Recently, plasmonic photothermal therapy (PPPT), which consists in the transduction

of light into heat by plasmonic nanoparticles to generate rapid localized heating, has emerged

as a potential, minimally invasive alternative for cancer treatment with reduced side

effects.(34) As described in Chapter 2, electromagnetic waves incident on a plasmonic

nanoparticle at the surface plasmon resonance (SPR) frequency induce a resonance oscillation

on the particle surface.(34) These nanoparticle plasmons can decay by two mechanisms:

radiative damping (scattering) and nonradiative dissipation.(55) The absorbed energy is

converted into heat by a nonradiative mechanism through consecutive photophysical process

that have been studied by ultrafast dynamics.(89) First, the generated surface plasmon

resonances transfer their energy to the electrons through Landau damping. These energized

electrons undergo rapid electron-electron scattering and within a few femtoseconds establish a

nonequilibrium “hot” electron distribution, usually called hot electrons, characterized by an

elevated temperature. These hot electrons then thermalize with the lattice via electron-phonon

interactions on a picoseconds time scale. The heat is then dissipated to the surrounding

medium via phonon-phonon coupling within hundreds of picoseconds, resulting in an increase

in temperature of the surrounding medium by tens of degrees.(55,90)

The key factors that led to the PPTT efficacy include: (i) sufficient tissue penetration

of the excitation light, (ii) large light–absorption cross section of the agents in the excitation

wavelength,(iii) high photothermal conversion efficiency, and (iv) enough accumulation in

the target tissue. The excitation light wavelength has to be in a region with maximal

penetration through tissue. It is known that hemoglobin and water have their lowest

65

absorption coefficient in the near infrared region (NIR) region, more specifically from 650 to

900 nm (Figure 3.1).(8) The other region of transparency has been described to be between

1000 and 1350 nm.(91)

Figure 3.1 - The near infrared region (NIR) window for in vivo applications, showing the region of minimal

light absorption by hemoglobin and water.

Source: WEISSLEDER et al.(8)

Second, the plasmonic nanoparticles must have large light–absorption cross section in

the excitation wavelength. As discussed in Chapter 2, gold-based nanoparticles present

interesting optical absorptions, which can be easily tuned to NIR by modifying their structural

characteristic such as geometry, size, and shape.(92) Therefore, the absorption in the NIR

region makes gold nanorods, nanoshells, and nanomatryoshkas suitable for in vivo

applications and efficient light-to-heat converters due to their large absorption cross-

section.(10, 65) Further, gold nanorods, nanoshells, and nanomatryoshkas present high

photothermal transduction efficiency, which represents the ability of a nanoparticle in

converting absorbed light into a temperature increase of its surrounding medium, and these

systems have proved to have high potential for photothermal therapy.(10, 55)

Nanostructures NIR tranducers agents present several advantagens over conventional

phothothermal agents. Besides the fact that their properties can be adjusted in the systhesis

process by controlling their geometry and composition,(54) gold nanomaterials have shown

good chemical/thermal/photo stability and low cytotoxicity.(58) Their strong absorption

cross-section also allows effective laser therapy using lower energies.(34) Moreover, the

facility to modify the gold surface allows the incorporation of drugs and biologic molecules

for applications in drug-delivery and therapy.(93-94) Additionally, they can provide high-

contrast images of cancer cells using a variety of optical techniques, including dark-field

66

microscopy,(44) reflectance confocal microscopy,(45) one- or two-photon fluorescence

imaging,(46) surface enhanced Raman scattering (SERS),(47-48) and photoacoustic

imaging,(49) which open opportunities for fabrication of theranostic platforms. Furthermore,

nanomaterials have demonstrated extension of circulating half-life and higher passive

accumulation in tumor sites (EPR effect) when compared to small molecules based

systems.(57,95)

Gold nanoshells were the first nanoparticle used to demonstrated PPTT by Halas and

co-workers in 2004.(96) Complete regression of the tumor after 10 days of gold nanoshell

photothermal treatment has reported, and after 90 days post-treatment, all mice remained

healthy and free of tumors, while tumors in control groups continued to grow rapidly.(7) The

potential of gold nanoshells for photothermal therapy in tumor together with their low

cytotoxicity have led to their current use in clinical trials for head and neck cancer

treatment.(97-98) Recently, Ayala-Orozco et al. demonstrated that the tumor uptake of gold

nanomatryoshkas was higher than gold nanoshells, and, consequently nanomatryoshkas

presented an improved photothermal therapy efficacy. They attributed this enhancement to the

more appropriate diameter of nanomatryoshkas (~100 nm) when compared to nanoshells

(~150 nm).(53)

The use of gold nanorods for PPTT applications has also been widely explored in the

last years.(92) Dickerson et al. demonstrate that PEGylated-AuNRs can be efficiently used for

in vivo PPTT treatment of deep-tissue malignancies.(9) AuNRs have also been proved to be a

non-invasive and efficient procedure for the treatment of lymph node metastasis.(99) Silica-

coated AuNRs has also been demonstrated to be a great platform to be used as a dual probe

for imaging and cancer therapy.(100)

The mechanism of cell death due to PPTT using gold nanoparticles has been

investigated. According to recent studies, it is clear that one single mechanism may not be

sufficient to describe the effects of PPTT.(90) Apoptosis and/or necrosis and sometimes also

oncosis have been reported. The observation of different cellular responses to the PPTT

results from variation in methods of nanoparticle delivery, dosing, and irradiation

conditions.(90) Nanoparticle location also influences the toxicity of photothermal heating. For

example, Tong et al observed that the cells with internalized AuNRs required 60 mW laser

intensity to cause cell damage, while for surface-bound nanorods only 6 mW was

necessary.(101) In this case, cell death was attributed to the disruption of the cell membrane.

The laser intensity during PPTT also plays a significant role in the cell death mechanism.

According to Li et al, for cells irradiated with a femtosecond pulsed scanning laser of 13.9

67

and 9.5 W/cm2 the death was caused only by apoptosis. Increasing the laser power to 27.8

W/cm2 (10 scans) also induced apoptosis, while 30 scans induced necrosis, and 55.6 W/cm

2

resulted in necrosis after only 1 scan.(102) To better understand the mechanism of cell death,

Chen et al monitored the EMT-6 breast cancer cells treated overnight with gold nanorods

during PPTT in real-time. The nanorods were located in the lysosomes, and irradiation with a

femtosecond pulsed laser exploded them; however, cell death was not immediate. According

to the authors, photothermal treatment caused the formation of 10 μM cavities, leading to

rupture of the plasma membrane.(103)

3.1.3 Combined therapies

The heating caused by phototherapy may also increase susceptibility of cancer cells to

other treatments. Several studies have reported that a synergic toxicity effect resulted from the

combination of gold nanomaterials and chemotherapeutic drugs in PPTT.(90) For example,

Chen et al developed doxorubicin-conjugated gold nanorods for combined thermal therapy

and chemotherapy in vivo.(104) The therapeutic effect and tumor inhibition of the

multifunctional platform with NIR irradiation was more pronounced than free doxorubicin or

only gold nanorods + laser. In another study, gold nanorods and doxorubicin co-loaded

polymersomes were developed.(105) It was also found that co-therapy significantly improved

therapeutic efficacy compared with chemo or photothermal therapy alone.

Many studies have reported the use of gold nanorods and photosensitizers for a

combined effect of PPTT and PDT.(92) When chlorin-p6, a widely used photosensitizer, is

conjugated with gold nanorods, the production of singlet oxygen increases significantly.(106)

This effect has been attributed to the coupling with the plasmons of the AuNRs. Moreover,

the uptake of chlorin-p6 was more efficient in the conjugate compared to free chlorin-

p6.(106) In a similar platform, Wang et al. developed a multimodal therapy using gold

nanorods conjugated with an aptamer, and the photosensitizer chlorin-e6.(107) They showed

that the aptamer changes its structure upon irradiation and release the photosensitizer for

production of singlet oxygen, resulting in an enhanced therapeutic effect.(107) Kuo et al

proposed the conjugation of gold-based materials with the hydrophilic photosensitizer,

indocyanine green.(108) They showed that the combination of PPTT and PDT proved to be

more efficient in killing cancer cells compared to both treatments alone. They also described

an enhanced photostability of the indocyanine green after conjugation.

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3.1.4 Cell membrane coated nanomaterials

Although much progress has been made in applications of nanomaterials for

diagnostic and therapy, significant clinical advances have not been observed. One of the main

reasons for this failure is the low accumulation rate of the nanoparticles in the target tissue,

which depends on their ability to escape the immune system, cross the biological barriers and

accumulate at target tissues.(12)

It is known that nanomaterials can accumulate in the tumor by a passive or active

mechanism. The passive accumulation is attributed to the enhanced permeability and retention

effect (EPR).(57) However, the variety of vascularization degree and permeability of the

tumors together with the relative short blood circulation time of nanomaterials have limited

this strategy.(20) In the active accumulation, the nanomaterials are functionalized with

specific biomolecules, such as antibodies,(109) peptides,(110) aptamers,(111) which

specifically recognize the receptors overexpressed on cancer cells membranes.

Recently, it has been shown that the ability of the biomolecules-functionalized

nanomaterials to target the cancer cell can be damaged when they are in a biological

environmental. Salvati et al. demonstrated that Transferrin-functionalized nanoparticles, a

protein that recognizes receptors of transferrin overexpressed in cancer cells, lost their

specificity in the biological media.(112) This effect has been attributed to the interaction of

the nanocomposite with proteins of the media, called opsonization, which can form a layer of

unspecific proteins around the nanomaterial, known as corona.(113) Similarly, Xiao et al

demonstrated that the functionalization of gold nanorods with cRGD peptides, which

recognized specifically αvβ3 integrin, an important biomarker for cancer cells, did not provide

a significant advantage in the in vivo tumor accumulation.(114)

The functionalization of nanoparticles with polyethylene glycol (PEG) has been

widely explored to extend the in vivo blood circulation time of the particles and improve their

passive and active accumulation. The addition of PEG to the nanoparticle surface can avoid

the adhesion of opsonins proteins present in the blood serum, increasing their blood

circulation time and consequent tumor accumulation.(14) PEGylation also increases the

nanoparticle stability in buffer and serum due to the hydrophilic ethylene glycol repeating

units.(14) Although much progress has been made, the accumulation in the target region is

still low, reducing the therapeutic effects of nanoparticles and increasing the risk of side

effects. Also, some evidences of the immunological response anti-PEG have been

reported.(15)

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Overcoming the immune system barriers and enhancing the accumulation of the

nanoparticles in the tumor are the major challenges in nanomedicine today.(115) These

problems have inspired the development of new functionalization strategies. Among them, the

coating with natural cell membranes has been shown to be an interesting alternative to

camouflage the particles and extend their blood circulation time. Hu et al showed that red

blood cell membrane (RBC)-coated polymeric nanoparticles present longer blood circulation

time than the PEG-coated ones (Figure 3.2).(16) More recently, the authors showed

evidences that the CD47 proteins, a biomolecule capable to inhibit the phagocytosis and

modulate the immunological response, are transferred to the nanoparticle’s surface and get

exposed, allowing them to perform molecular interactions and reducing their susceptibility for

being captured by macrophages.(17) The ability to transfer to nanomaterials

immunomodulatory proteins including CD47 at an equivalent density to that of natural cells

has been described as one of the most important characteristic of the cell membrane approach.

Figure 3.2 - Schematics of the synthesis of the Red blood cell (RBC) membrane-coated polymeric PLGA

nanoparticles.

Source: HU et al.(16)

RBC membrane coating has also been used to reduce the phagocytic uptake and

extend the blood circulation time of gold nanoparticles,(116) gold nanocages,(117) iron

oxide,(118) and upconversion nanoparticles.(119) Similarly, leukocytes cell membrane-

coated porous silica nanoparticles have shown lower tendency to be eliminated by

immunological system.(12) Ding et al developed erythrocyte membrane-coated upconversion

nanoparticles and photosensitizer for NIR-triggered photodynamic therapy applications.(119)

Erythrocyte ghosts have been used to encapsulate iron oxide for magnetic resonance imaging

70

applications.(118) Piao et al also showed that RBC membranes-coated gold nanocages exhibit

significantly in vivo blood retention and circulation lifetime compared to

poly(vinylpyrrolidone) coated ones.(117) As a result, this system demonstrate enhanced

tumor uptake when administered systematically, allowing a much more effective

photothermal therapy treatment in vivo. These new biomimetic systems have demonstrated

enhanced tumor uptake with potential for more effective applications in medicine.

The cell membrane coating strategy has been extended to cancer cells. Fang et al

showed that the coating of PLGA nanoparticles with cells membranes derived from MDA-

MB-435 cancer cells allows particle functionalization with multiple membrane-bound tumor-

associated antigens.(18) These systems possess the same adhesion domains of its source

cancer cells, responsible for multicellular aggregate formation in tumors, and have

demonstrated increasing specificity for cancer cell targeting.(18) This strategy allows

mimicking the complex functions of the cells without using engineered antibodies or

aptamers, which could have high cost and, as discussed early, may not be effective to target

cancer cells. Also, the cancer cell membrane coating strategy may also be explored for cancer

immunotherapy.(19) Therefore, this emerging top down approach of functionalization cannot

only increase the in vivo circulating time of the nanoparticles and, consequently, their

accumulation in the tumor, but also can add new functionalizations and properties to

nanosystems.

3.1.5 Cell membrane vesicles as carriers

Natural cell membrane vesicles have also shown great potential as drug delivery

systems. The use of reconstructed membranes from stem cell as platform for drug-delivery

(20) has been reported as well as the loading of indocyanine green into nano-sized vesicles

derived from erythrocyte cells for combined optical imaging and phototherapy of

tumors.(120) Capsules derived from erythrocytes have exhibited many favorable properties

such as efficient drug loading, biocompatibility, and prolonged blood circulation time.(121-

122)

Similarly, cancer cells-derived vesicles have emerged as new drug delivery system

with increasing specificity. Methotrexate-loaded tumor cells microparticles have proved to

effectively deliver therapeutic agents to tumor cells in murine tumor models, killing them

without typical side effects associates with the drug.(123) A recent study also have reported

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the fabrication of a multifunctional chemo- and photo-thermal agent composed of folic acid

(FA)-modified, multi-layered, hollow microcapsules loaded with gold nanorods and the anti-

cancer drug doxorubicin.(104)

3.1.6 -Lapachone

-Lapachone (β-Lap) is an ortho-naphthoquinone with molecular weight of 242.27 Da

(Figure 3.3) that has attracted significant interest in the last years. It is a plant-derived

anticancer agent, first isolated from the bark of Lapacho tree (genus Tabebuia) in South

America rainforests.(124) Currently, β-Lap is produced through cyclization of lapachol in

sulphuric acid.(125) One of the most advantages of β-lap is the fact that its cytotoxicity is

activated by NADP(H):quinine oxidoreductase (NQO1), an enzyme highly expressed (up to

20x) in many human cancers, such as lung, prostate, breast, and pancreas.(126) NQO1

detoxifies harmful quinines by catalyzing two-electron reduction of the quinines to

hydroquinones using NADH or NAD(P)H as the electron source. It has been suggested that

NQO1 causes a futile redox cycle between β-Lap and its two electron reduced hydroquinone

form, leading to a generation of reactive oxygen species, Ca2+

-dependent DNA damage,

hyperactivation of poly ADP polymerase-1, and decrease in the NAD+ and ATP, which

results in cell death.(127-128) Its anti-cancer effect has been described to be independent of

p53, caspase, and cell cycle status, which can minimize drug resistance.(124) Therefore, β-

Lap presents very low toxicity to normal cells and can be used as an effective and selective

antitumor agent.

Figure 3.3 - Chemical structure of -Lapachone.

Source: SIGMA…(129)

The lethal concentration of β-lap has been shown to be around 1 and 30 µM, and for

most of cancer cells lines, this value is around 1 to 5 µM.(130) However, its poor solubility,

low stability in circulation, short blood circulation time, and poor specificity led to low tumor

72

accumulation and have limited its therapeutic applications.(124) To overcome these problems,

initial studies have focused on increasing the aqueous solubility of β-Lap through

complexation with hydroxypropyl-β-cyclodextrin.(131) However, the fast dissociation of this

complex, low tumor accumulation and recent observations about cyclodextrin side effects

(128) have motivated the search for new alternatives.

Recently, substantial efforts have focused on nanostructured systems. The

encapsulation of β-Lap inside polymeric nanostructures has increased its stability while

maintaining its NQO1-dependent cytotoxicity.(128, 132) Also, new properties such as optimal

size, longer blood circulation time, diffusion-based release kinetics, and multifunctionality

can be achieve using a nanostructure platform. Zhang et al coencapsulated paclitaxel, one of

the most effective broad-spectrum chemotherapeutic agent, together with β-Lap into

PEG−PLA micelles.(124) They demonstrated a strong synergistic cytotoxicity effect against

cancer cells, including pancreatic cancer cells. Conjugation of β-Lap with graphene oxide

(133) and gold nanoparticles (134) have also been demonstrated. These β-lap nanoconjugates

have demonstrated selective cytotoxicity, and represent a potential treatment for a

multifunctional strategy against cancer.

3.1.7 Toxicity of nanomaterials

The potential applications of nanoparticles in medicine are highly dependent on

toxicology studies to determine their stability and effects in a biological medium.(135) The

toxicity of gold-based nanomaterials has been described to be dependent on several

parameters such as size, shape, and surface functionalization.(92) For gold nanorods, the high

concentrations of cytotoxic cetyltrimethyl ammonium bromide (CTAB) used in the synthesis

and the difficulty to displace it efficiently without compromising the properties of particles is

a key barrier to guarantee their efficiency and safety.(136) The surfactant may internalize into

the cells with and without the AuNRs, damaging the mitochondria, and inducing

apoptosis.(137) Many strategies have been developed to overcome this problem. For example,

due to the high binding affinity of sulfur-metal bond, thiol-containing molecules have been

extensively used to replace CTAB molecules. One of the most used methods consists of

replacing CTAB with thiolated polyethylene glycol (PEG),(138-140) because PEG provide

high stability in biological media and may extend the blood circulation time of

nanomaterials.(141)

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The influences of the aspect ratio and the surface functionalization in the toxicity and

uptake of gold nanorods have also been investigated.(137) Qiu et al showed that although the

cellular internalization is highly dependent of the aspect ratio and surface modification, only

the functionalization contributed significantly to the toxicity. Moreover, the formation of a

protein around the nanorods have significantly reduced their toxicity.(142) Adura et al.

showed that a conjugate containing AuNRs and CLPFFD peptide, that recognizes toxic β-

amyloid aggregates present in Alzheimer’s disease, has less influence on the cell viability

compared to the regular AuNRs coated with CTAB.(143) The protein corona approach has

been demonstrated to reduce the toxicity of AuNRs.(142) A comparative analysis of the

impact of fetal bovine serum (FBS)-AuNRs and CTAB-AuNRs on the cytoplasmic membrane

structure revealed that CTAB-AuNRs aggregates on the membrane of A549 cells (human

lung adenocarcinoma epithelial cell line), destroying it. For FBS-AuNRs, the presence of the

protein corona facilitated the protein receptor-mediated internalization and translocation of

AuNR to endo-/lysosomes, without causing dramatic effects on the membrane structure.(142)

Although much progress has been made regarding nanotoxicology, the variety of

nanomaterials, functionalizations, size and shape, combined with the lack of standardization

in the assays, have led to some inconclusive results. Lison, D. et al have systematically

revised studies related to cytotoxicity of silica nanomaterials to show the gaps and the need

for standardization studies in that area.(135)

3.2 EXPERIMENTAL SECTION

3.2.1 Materials

All reagents were used without any further purification and the solutions were

prepared using ultrapure water (18.2 MΩ cm). All glassware were cleaned with aqua regia

and thoroughly washed with distilled and Milli-Q water. Cetyltrimethylammonium bromide

(CTAB), silver nitrate (AgNO3), tetrachloroauric acid (HAuCl4), sodium borohydrate

(NaBH4) e ascorbic acid were purchased from Sigma-Aldrich. β-Lapachone (β-lap) and

poly(ethylene glycol) methyl ether thiol (mPEG-SH) average Mn 5,000 were also obtained

from Sigma-Aldrich.

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3.2.2 Gold nanorods (AuNRs) synthesis

Gold nanorods (AuNRs) were synthesized by the seed-mediated method in presence of

the surfactant CTAB.(72) Briefly, the seeds were prepared by adding 250 µL of

HAuCl4.3H2O 0.01 mol L-1

in 7.5 mL of CTAB aqueous solution 0.1 mol L-1

under gentle

stirring. Next, 600 µL of iced cold NaBH4 0.1 mol L-1

was added and the system was kept

under vigorous stirring for 10 min. The color of the solution changed from yellow to pale

brown. The solution was kept at 25ºC for about 2 hours before using. The growth was

performed by adding 4 mL of 0.01 mol L-1

HAuCl4.3H2O in 47.5 mL of 0.1 mol L-1

CTAB

aqueous solution, followed by 600 µL of 0.01 mol L-1

AgNO3 and 480 µL of 0.01 mol L-1

ascorbic acid solutions. The color of the system changed from dark yellow to transparent,

indicating the reduction of the Au3+

to Au+. Finally, 70 µL of the seed solution previously

prepared were added and the system was kept under very gentle stirring for 20 min. The

system was left at room temperature and protected from light for 4 h, and the supernatant was

washed by centrifugation at 10000 g for 5 min. The suspension was cooled at 4ºC to

crystallize the excess of CTAB and centrifuged at 2000 g for 2 min to remove the crystals.

The resulting nanorods were subsequently functionalized with poly(ethylene glycol)

methyl ether thiol (mPEG-SH). For this, mPEG-SH was mixture with the AuNRs in a final

concentration of 200 µmol L-1

and the system was sonicated for 30 minutes. The particles

were shaken for more 12 hours at room temperature and then centrifuged (10000 g, 5 min) to

remove the excess of mPEG-SH and CTAB. The concentration of the resulting PEG-AuNRs

for the experiments was standardized by their optical density at 750 nm.

3.2.3 Cell culture

Human lung adenocarcinoma epithelial (A549) and rat hepatocarcinoma (HTC) cells

were acquired from Rio de Janeiro Cell Bank (BCRJ). Human hepatoma (HepaRG) cells were

kindly supplied by Dra. Natalia Inada from the Optics Group, Physics Institute of Sao Carlos,

University of Sao Paulo, Brazil. All the cell culture reagents were previously autoclaved and

the procedures were performed in a laminar flow hood using sterile materials. All the cells

lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Vitrocell, Campinas,

Brazil) supplemented with 10% of Fetal Bovine Serum (Vitrocell, Campinas, Brazil) and

maintained at 37 °C and in 5% CO2 in a humidified incubator. Cells viability was investigated

75

always before the assays (data not shown) according to the trypan blue (Sigma-Aldrich, USA)

exclusion assay, showing viability higher than 95%.

3.2.4 Extraction of A549 cancer cell membrane (CM)

The cancer cell membranes were isolated and purified by ultracentrifugation based on

a previous method proposed by Lung et al (144) and reconstructed as vesicles by extrusion.

Briefly, three cell culture flasks of 160 cm2 containing about 2x10

7 A549 cells/each were

rinsed in PBS and removed from flasks using trypsin solution 2.5 g/L with EDTA (Vitrocell,

Campinas, Brazil). The cells were washed there times by centrifugation (1000 g, 5 min) in

PBS, and lysed by incubation in a hypotonic buffer (10 mM Trisbase, 1.5 mM MgCl2, 10 mM

NaCl, pH 6.8) for 5 min at 4ºC. The lysed cells were centrifuged (300 g, 5 min), resuspended

in the gradient buffer (0.25 M Sucrose, 10 mM HEPES, 100 mM Succinic acid, 1 mM EDTA,

2 mM CaCl2, 2 mM MgCl2, pH 7.4), and homogenized by 70 strokes (1900 rev/min) using a

homogenizer (Glass homogenizer VIRTUS PII), followed by centrifugation at 10000 g for 10

min at 4ºC to remove cell debris. The supernatant was collected and centrifuged at 150000 g

for 1h20 using an ultracentrifuge Optima MAX-XP (Beckman Coulter, USA) at 4ºC. The

pellet, containing the membranes, was dispersed in 1 mL of PBS containing Complete

Protease Inhibitor (Roche Diagnostics, Mannheim, Germany) and stored at -80ºC.

3.2.5 A549 cancer cell membrane (CM) characterization

The membrane was characterized by sodium dodecyl sulfate polyacrylamide 10%

gel electrophoresis (SDS-PAGE). The standard BenchMark Protein Ladder (Invitrogen) was

used as reference for mass determination and the gel was fixed and stained with silver nitrate

(ProteoSilver kit, Sigma-Aldrich). The quantitative determination of protein in the cell

membrane was performed by the QuantiPro BCA Assay Kit (Sigma-Aldrich), a colorimetric

method for the determination and quantification of total proteins based on the capacity of the

proteins to reduce Cu2+

to Cu+ in an alkaline medium. The purple color results from the

interaction of the bicinchoninic acid (BCA) with the ion Cu+ and the absorption at 562 nm

exhibited by the complex is approximately linear with the increasing of the protein

concentration in a wide range (20-2000 µg/mL). The procedure was performed according to

the described at the assay’s protocol. First, a calibration curve was obtained using known

76

concentrations of bovine serum albumin (BSA) (Figure 3.6) and then different concentrations

of the cell membrane were measured and the concentration determined from the calibration

curve. All the absorbance measurements were performed in a 96-well plate using a

SpectraMax M3 microplate reader (Molecular Devices).

The lipid composition of the cell membrane was analyzed by Iatroscan TLC/FID

(Iatroscan MK-VI, Iatron, Japan), which combines thin-layer chromatography (TLC) and

flame ionization detection (FID) using hydrogen flow of 174 mL/min and air flow 2000

mL/min. It has been shown to be an important technique in lipid determination.(145) The

lipids standards HC (aliphatic hydrocarbons), WE/SE (ester), TAG (triglycerides), FFA (free

fatty acids), ALC (aliphatic alcohol free), ST (sterol), AMPL (mobile polar lipids in ketone)

and PL (phospholipids) were obtained from Sigma-Aldrich and all other chemicals and

solvents were of analytical grade. For this analysis, the A549 cell membranes were

resuspended in chloroform after ultracentrifugation using the concentration as previously

described. Before the Iatroscan analysis, the samples were homogenized in an ultrasound bath

at 4ºC for 15 min. The lipids samples were resolved in three steps, which involve subsequent

elution stages with increase solventes polarity. These are: solution 1 (98.95% hexane, 1%

diethyl ether and 0.05% formic acid), solution 2 (79% hexane, 20% diethyl ether and 1%

formic acid), solution 3 (100% acetone) and solution 4 (50% of chloroform, 40% methanol

and 10% water).

3.2.6 A549 cell membrane-loaded β-lapachone (CM-β-Lap)

The cell membrane with a protein concentration of 774.9 µg/mL was sonicated in an

ice bath sonicator at 4ºC for 15 min. Then, -Lap (0.07 mg, ~0.3 mmol L-1

) was added to the

system and sonicated in an ice bath sonicator at 4ºC for 15 min, followed by gentle shaken for

2 h at 4ºC and protected from light. The system was extruded through 200 nm polycarbonate

membrane using a miniextruder (Avanti Polar Lipids), and dialyzed against 0.2x PBS buffer

at 4ºC and protected from light for 3 hours to remove the remnants from cell-membrane

extraction and the excess of -Lap. The result suspension was collected and kept at 4ºC.

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3.2.7 A549 cell membrane-loaded β-lapachone-coated PEG functionalized gold nanorods

(CM-β-Lap-PEG-AuNRs)

The CM/AuNRs ratio was tested and optimized in order to have uniformity, surface

stability and reproducibility. After dialysis, CM-β-Lap suspension was extruded 11 times

through 200 nm polycarbonate membrane using a miniextruder (Avanti Polar Lipids),

followed by more 7 times in 100 nm. The size and distribution of the extruded membrane

were evaluated by Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Marvern). 500 µL of

this suspension was added to 2000 µL of PEG-AuNRs (optical density at 770 nm = 0.5) and

extruded 7 times thought polycarbonate membrane of 100 nm. The system was centrifuged

(5000 g, 10 min) to remove the excess of membrane or β-Lap and the particles were

ressuspended in 1x PBS.

β-lap loading concentration in the final system was determined by destabilization of

the cell membrane to release the anticancer agent. To this, CM- β-lap-PEG-AuNRs were

diluted in ethanol and sonicated for 30 min. The particles were then centrifuged at 10000g for

15 min and the supernatant spectrum was measured using an UV−VIS spectrophotometer

(Hitachi U-2900), and the appropriate baseline. The concentration was calculated using the

extinction coefficient calculated from the calibration curve (Figure 3.5).

For comparison in the cytotoxicity and phototherapy assays, we also synthesized A549

cell membrane-coated PEG-functionalized gold nanorods (CM-PEG-AuNRs), i.e., without β-

lap, using the same procedure as described for CM-β-Lap-PEG-AuNRs. The concentration of

free PEG-AuNRs, CM-PEG-AuNRs and CM-β-Lap-PEG-AuNRs was set using the same

optical density (OD) of the longitudinal plasmon peak.

3.2.8 Photothermal therapy

The optical properties of the CM--Lap-PEG-AuNRs were analyzed and compared

with the PEG-AuNRs. 700 µL of each system (OD750 nm = 0.59) in a 3 mL polystyrene cuvette

were irradiated using a cw 808 nm diodo laser with elliptical beam of diameter ~ 1.5 mm x

3.0 mm (iZi, LASERline). The power at the distance of irradiation was measured and adjusted

to have a power density of approximately 1.5 W/cm2. The temperature was monitored using

an optical thermometer (FOT Lab Kit Fluoroptic Thermometer, LUXTRON). The initial

temperature was the room temperature in the laboratory (~ 23ºC).

78

The release of -Lap was determined based on previous methods.(128) Briefly, the

final CM--Lap-PEG-AuNRs sample was divided in four aliquots (400 µL each), irradiated

with the laser with a power density of approximately 1.5 W/cm2

at different times (5, 10, 15,

20 min), and centrifuged immediately to separate the released -Lap from the particles at

(5000 g, 10 min at 4ºC) using a Microcon centrifugal filters (MW cutoff = 3 kDa). Typical

absorbance of the -Lap at 257 nm was measured in the resulting filtrate and used to calculate

the release concentration based on the calibration curve (Figure 3.5).

For the in vitro photothermal analysis, 5x104 A549 cells were seed in a 96-well plate.

After 24 h, the media was removed and 140 µL of media containing of PBS (control), CM--

Lap-PEG-AuNRs or CM-PEG-AuNRs were added to each well in triplicate. We use the

absorbance at the maximum longitudinal peak to standardize the concentrations of both

systems. The final OD at 750 nm in cell culture media in both systems was 0.3. The cells were

incubated for 4 hours 37 °C and 5% CO2 and washed twice with PBS. Finally, 100 µL of

media were added to each well and the cells were irradiated using the same cw 808 nm diodo

laser with elliptical beam of diameter ~ 1.5 mm x 3.0 mm (iZi, LASERline) and power

density was also adjusted to have approximately 1.5 W/cm2. After exposure to the laser light

for 10 min, the cells were incubated for additional 2 h at 37 °C and 5% CO2, followed by

staining with 0.4% trypan blue (Sigma-Aldrich) to evaluate the viability. The images were

taken using an inverted light Zeiss microscope.

3.2.9 Characterization

The optical properties of the PEG-AuNRs before and after conjugation with CM--

Lap were characterized by Ultraviolet-Visible-Near Infrared (UV-VIS-NIR) spectroscopy

(Hitachi U-2900). Their size, distribution and morphology before and after laser irradiation

were analyzed by Field-emission Scanning Electron Microscopy (FEG-SEM). For this, silicon

substrates (P-type/boron-doped silicon, Sigma Aldrich) were cleaned with isopropanol,

ethanol and water in an ultrasound bath (2 min each) and dried in a stream of nitrogen gas.

The samples were diluted in water, drop-cast onto the substrate and allowed to dry in ambient

conditions for 12 h. Field-emission SEM imaging was obtained with a Zeiss Sigma VP FEG-

SEM at 3 kV in high vacuum mode.

Dynamic Light Scattering (DLS) was used for characterizing the size and aggregation

state of nanomaterials in situ, i.e., in suspension. DLS measures the light scattered from a

79

laser that pass through the colloidal suspension and by analyzing the modulation of the

scattered light intensity as function of time, the hydrodynamic size and agglomerates can be

determined. Simply, the technique is based on the Brownian motion of the particles in

suspension. Therefore, larger particles will diffuse slower than the smaller ones and the time

dependence of the scattered light to produce a correlation function can be mathematically

linked to the particle size. The analyses were performed in triplicate at 25ºC using a Malvern

Nano-ZS spectrometer (Malvern Instruments, UK).

Zeta Potential was used to determine the surface charge of nanoparticles in

suspension. Nanoparticles have a surface charge that attracts a thin layer of ions of opposite

charge to the particle surface, forming the double layer, which diffuses throughout the

suspension with the particle. The electric potential at the boundary of the double layer is

known as the zeta potential of the particles, whose values typically range from -100 to +100

mV. The magnitude of the zeta potential is an indicative of the electrostatic colloidal stability.

The analyses were performed in triplicate at 25ºC using a Malvern Nano-ZS spectrometer

(Malvern Instruments, UK) and the results are presented as mean ± standard derivation

resulting from three different measurements.

3.2.10 Isothermal Titration Calorimetry (ITC)

ITC has emerged as an useful technique to investigate the thermodynamic interaction

between nanoparticles and molecules.(146, 147) Previous studies have shown the use of ITC

to evaluate the capacity of nanoparticles to avoid protein adsorption, which is a relevant

property to decrease the opsonization and extend the blood circulation time of the

nanomaterials.(148) In this study, interactions between BSA, a major representative globular

protein present in the serum, and PEG-AuNRs and CM--Lap-PEG-AuNRs were evaluated.

ITC experiments were performed in a MicroCal ITC200 (Malvern Instruments) at 25 °C with

a continuously stirred at 250 rpm by titrating BSA protein solutions (10 µmol L-1

) into the

sample cell containing the nanoparticles. 2 µL of BSA were titrated 18 times with 180

seconds between injections. As a comparison, we also performed the titration of BSA solution

into CTAB-coated AuNRs. However, since CTAB-AuNRs presented low stability in buffer

solution, CTAB/PEG-AuNRs, which consisted in the PEGylation of the CTAB-AuNRs using

a lower concentration of mPEG-SH, just enough to keep the particles stable in 0.1xPBS were

used. The zeta potential of CTAB/PEG-AuNRs was around +18 mV. The concentration of the

80

three nanoparticle systems was kept about the same (OD at 750 nm = 0.6) and all samples

were in 0.1xPBS buffer. The heat of dilution of the protein was performed by titrating BSA

into buffer at the same concentrations. No quantitative analysis was performed due the

difficult to determine exactly the molar concentrations of the particles.

3.2.11 Cellular Uptake

The cell uptake was evaluated using different strategies. First, the CM-PEG-AuNRs

were labeled with fluorescent dye molecules for optical tracking. The CMs were stained with

CellMask Deep Red plasma membrane stain (Life Technologies) with excitation/emission

wavelengths of 649/667 nm. The system was incubated for 30 min at 4ºC and protected from

light. The next steps of dialysis and conjugation with PEG-AuNRs were performed as

described previously. It is important to note that at the end of the conjugation, the CM-PEG-

AuNR system is centrifuged, which also allows to remove the excess of the dye molecules.

Next, A549 cells were seeded in a µ-Dish 35 mm (Ibidi) and cultured for 24 h at 37ºC and 5

% CO2, following by changing the media by fresh media containing CellMask-labeled CM-

PEG-AuNRs and incubated for 4 h. As a control and since no information about CellMask

stain is available, we observed the effect of free CellMask stain incubated with A549 cells in

the same conditions. The cells were rinsed twice with PBS and fixed with 4% formaldehyde

in PBS for 10 min, followed by rinsing with PBS. The cell nuclei were stained by incubating

the cells with 5 mg/mL Hoechst 33342 dye solution (Sigma-Aldrich) for 15 min protected

from light. Cells were washed in PBS and imaged using a Zeiss LSM 780 Confocal

Microscope - Inverted Microscope.

The cellular uptake was also evaluated by UV-VIS-NIR spectroscopy.(149) In this

case, cells were plated in 6 well plate and, after 24 h, the media was replaced with fresh media

containing PEG-AuNRs or CM--Lap-PEG-AuNRs with a final OD at 750 nm of 0.3. At

each point time, the cells were rinsed three times with PBS to remove the excess of particles

and detached from the wells using trypsin solution 2.5 g/L with EDTA. Next, the cells were

centrifuged, resuspended in 1 mL of PBS buffer, and counted using a hemocytometer. The

cells were centrifuged again and resuspended in hypotonic buffer (10 mM Trisbase, 1.5 mM

MgCl2, 10 mM NaCl), pH 6.8, for 5 min to lyse the cells, followed by sonication to break big

molecular aggregates. The absorption was normalized by the number of cells at each point

and is presented as media ± standard derivation of three measurements.

81

3.2.12 Cytotoxicity

The cytotoxicity of the CM--Lap-PEG-AuNRs was evaluated using the colorimetric

assays crystal violet (CV) and 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide

(MTT). The same doses of PEG-AuNRs, CTAB-AuNRs, CM and CM--Lap were also tested

in parallel for comparison. Three cell lines were used: the source cell (A549), another cancer

cell (HTC) and healthy human hepatoma (HepaRG). All the cell lines were cultured in 96-

well plate with a final volume of 200 µL of media for 24 h at 37ºC and 5 % CO2. After this

period, the media was changed by fresh media containing 5, 10 and 15 µL of the samples in

triplicate in a total of 100 µL in each well and incubated for 24 h at 37ºC and 5 % CO2.

In brief, for the MTT assay, the wells were washed twice with PBS and incubated with

100 µL of 0.5 mg/mL of MTT solution (Sigma-Aldrich, USA) for 4 h at 37 °C in a 5% CO2.

After, the MTT solution was carefully removed and replaced by 100 µL of dimethyl sulfoxide

(DMSO). Absorbance was measured at 570 nm on microplate reader SpectraMax M3

(Molecular Devices). For the crystal violet assay, after the incubation, the media was removed

and the wells were washed twice with PBS, using a multichannel pipette, followed by fixation

with ethanol 70% (10 min). The wells were washed twice with PBS and the cells were

incubated with 40 µL of violet crystal 0.5 % solution (Sigma-Aldrich) for 30 min at room

temperature. After that, the wells were washed at least 5 times with PBS 1% to remove the

excess of violet crystal and, finally, 100 µL of acetic acid 10% were added and incubated for

30 min. After 1 min of gentle shaking, the absorbance of each well was determined using the

microplate reader SpectraMax M3 (Molecular Devices) in 540 nm.

In both, the relative percentage of viable cells was calculated by considering the

negative control as 100% of viable cells. For all samples and concentrations, the experiment

was performed in triplicate and the assay was repeated two times for each period of

incubation, resulting in six measurements for each concentration. The results were analyzed

according with the media ± standard derivation of all measurements for each concentration,

and the values were statistically compared using the analysis of variance (ANOVA) and the

Turkey test (p < 0,05) conducted with OriginPro 8 software.

82

3.2.13 Detection of Intracellular Reactive Oxygen Species (ROS)

The generation of reactive oxygen species (ROS) was evaluated by 2',7'-

dichlorodihydrofluorescein diacetate (H2DCFDA), a non-fluorescent molecule that is

deacetylated by cytosolic esterases to a non-fluorescent compound dichlorodihydrofluorescein

(H2DCF), which in turn can be oxidized by ROS into a fluorescence molecule 2,7’ –

dichlorofluorescein (DCF). The cells were incubated with the samples for 2 hours, followed

by rising with PBS. The cells were then incubated with 5 µM of H2DCFDA in cell culture

media for 30 minutes at 37ºC and 5 % CO2, followed by washing with PBS. The fluorescence

was then measured at excitation/emission wavelengths of 485/530 nm using a microplate

reader SpectraMax M3 (Molecular Devices). The fluorescence intensity values were

normalized by the relative number of cells determined by Crystal Violet and are presented as

percentage considering the control as 100%.

3.3 RESULTS AND DISCUSSION

An overview on the different steps of the fabrication of the CM--Lap-PEG-AuNRs is

shown in Figure 3.4. First, the A549 cancer cell membranes (CM) were extracted and

purified by ultracentrifugation, followed by incorporation of the anticancer agent -Lap. The

gold nanorods were fabricated by the seed-mediated method in presence of CTAB and

covalently functionalized with mPEG-SH. The fusion of the PEG-AuNRs and CM--Lap

were performed by mechanical extrusion through 100 nm pore membrane.

83

Figure 3.4 - Schematic representation (not to scale) of proposed mechanism for the fabrication of the cancer

cell membrane-coated PEG-Gold nanorods and loaded with the anticancer agent -Lapachone

(CM--Lap-PEG-AuNRs). First, A549 lung cancer cells are isolated, lysed to remove the internal

content and purified by ultracentrifugation. -Lap is loaded on cell membrane structure and they

are extruded through 200 porous membrane (CM--Lap). In parallel, gold nanorods (AuNRs) are

synthesized by the seed-mediated method in presence of surfactant CTAB, followed by covalent

functionalization with mPEG-SH. The fusion of the resulting PEG-AuNRs and CM--Lap are

performed by mechanical extrusion through 100 nm pore membrane.

Source: By the author.

Figure 3.5 - a) Representative spectra of -Lap in water:ethanol (1:1) in different concentrations and b)

Calibration curve of -Lap at 257 nm made from triplicate of the spectra in different

concentrations.

Source: By the author.

300 400 5000.0

0.2

0.4

0.6

0.8

Absorb

ance / a

.u.

Wavelength / nm

0.1 M

1.0 M

2.0 M

5.0 M

10 M

15 M

20 M

30 M

0 5 10 15 20 25 30

0.0

0.2

0.4

0.6

0.8

Absorb

ance / a

.u.

Concentration / M

r2 = 0.9993

Abs257 nm

= - 0.0039 + 0.029 [Concentration]BA

84

0 300 600 900 1200 1500

0.0

0.2

0.4

0.6

0.8

1.0

Absorb

ance /

a.u

.

Protein concentration / mg mL-1

R2 = 0,997

Abs = 0,0119 + 5,008x10-4

[Protein]

Figure 3.6 - Calibration curve of BSA protein obtained from bicinchoninic acid (BCA) assay and used to

determine the protein concentration on the cell membranes vesicles (CM).

Source: By the author.

3.3.1 CM characterization

Cancer cell membrane vesicles (CM) were derived from A549 cancer cells. The

reasoning for choosing this cell line as a source lies in their metastatic ability.(150) Cell-

membrane-based systems have been shown to have the natural properties and some bio-

functions from their parent cells.(151) Taking advantage of the inherent homotypic binding

capability among tumor cells (18) and the metastatic characteristic of the A549 cells,(150)

their derived membrane vesicles seem good candidates for enhance drug and nanoparticle

delivery to the cancer and their metastatic sites.

The internal cell content was eliminated and the cells membrane was purified using

hypotonic lysing, mechanical membrane disruption and ultracentrifugation. The removal of

the internal components of the cell and the purification by ultracentrifugation is an important

step to eliminate the safety concerns regarding the genetic material of cancer cells.(18) The

vesicles were then formed by successive extrusion through 200 nm followed by 100 nm

porous polycarbonate membrane.

The qualitative analysis of the cell membrane proteins was performed by

polyacrylamide gel electrophoresis (SDS-PAGE), shown in Figure 3.7A. According to a

previous study, the resulting vesicles appear to retain the critical cell membrane proteins.(18)

The presence of specific and tumor-associate antigens from the source cancer cell line in the

derivative vesicles have been confirmed in a previous study performed by Fang et al.(18) The

quantitative analysis BCA of the proteins revealed a concentration of 774.9 µg/mL in the

sample, which was used to standardize the amount of CM in the synthesis and experiments.

85

The analysis of the lipid content of the CM by latroscan TLC/FID revealed that phospholipids

are the most abundant in the A549 cell membrane vesicle (Figure 3.8).

The next step was the loading of -Lap into the CM structure. -Lap is a very

promising anticancer agent due mainly to its specific toxicity, which is activated by

NADP(H):quinine oxidoreductase (NQO1), an enzyme overexpressed in many human

cancers, such as lung, prostate, breast, and pancreas.(126) The loading of -Lap is expected to

overcome the problems associate with this agent, such as short blood circulation time, low

stability in and poor specificity.(124) Several strategies have been developed for drug loading

in cell membrane vesicles such as lipid fusion,(152) endocytosis,(153) and hypo-osmotic lysis

method.(122) According to previous studies on fasudil-loaded erythrocytes nanovesicles, drug

loading was significantly higher at 4ºC compared to 25ºC and 37ºC.(122) Also, entrapment

efficiency was essentially unaltered when formulations were centrifuged at 5000 rpm, but an

increase in centrifugal force to 15000 rpm led to disruption of membrane and released of the

drug.(122)

The loading was performed by incubation of the CM with -Lap at 4ºC, followed by

extrusion through 200 nm porous membrane and dialysis. The hydrodynamic diameter

measured by dynamic light scattering (DLS) after extrusion through 200 nm membrane

confirms the formation of vesicles with an average diameter around 150 nm (Figure 3.7B).

Figure 3.7C shows a comparison of the UV-VIS spectra of the CM before and after the

loading of -Lap. The CM spectrum presents a small absorption around 260 and 220 nm,

which came from the proteins of the structure. After loading of the -Lap, the observed

spectrum represents a typical -Lap spectrum with a strong absorption peak at 257 nm.

Figure 3.7 - Characterization of the A549 cell membrane (CM). (A) Polyacrylamide gel electrophoresis (PAGE-

SDS) of the CM in duplicate. (B) Dynamic light scattering analysis of the CM--Lap after

extrusion extrusion through 200 nm porous polycarbonate membrane, and (C) UV-VIS spectra of

CM with/without -Lapachone.

Source: By the author.

0 100 200 300 4000

5

10

15

20

Inte

nsity %

Hydrodynamic diameter / nm

CM--Lap

200 400 600 800

Absorb

ance / a

.u.

Wavelength / nm

CM

CM--Lap

230

150

100

80

60

50

40

30

25

20

15

10

kDaA B C

86

Figure 3.8 - Iatroscan TLC/FID chromatograms of standard lipids and A549 cell membrane (CM) samples.

Source: By the author.

3.3.2 Synthesis of CM--Lap-PEG-AuNRs

To synthesize CM--Lap-PEG-AuNRs, gold nanorods were first prepared by the seed-

mediated method in presence of CTAB (72) and then covalently functionalized with mPEG-

SH. PEG coating is highly hydrated and this layer avoid interactions of the nanoparticle with

molecular and biological components of the biological media. Furthermore, as we will discuss

shortly, the removal of the CTAB is important to avoid cytotoxicity and destabilization of the

cell membrane vesicles.

A representative Scanning Electron Microscopy (FEG-SEM) image of PEG-AuNRs

and the histograms determined by measuring about 100 particles by SEM images are shown

in Figure 3.9 and revealed no aggregation of the system after the PEGylation process. The

calculated average aspect ratio (length/width) was approximately 3.5 and it is consistent with

the extinction spectrum of the PEG-AuNRs (Figure 3.10A). The spectrum is typical of

AuNRs, which exhibit two surface plasmon resonance (SPR). The band around 500 nm is due

the coherent motion of the conduction band electrons along short axes (transversal band),

while the band in the higher wavelength correspond to the oscillation long axes of the particle

(longitudinal band).(35, 59-60)

0 50 100 150 200

FID

/ m

V

Time / s

Standard

CMAMPL

PL

0 50 100 150

FID

/ m

V

Time / s

Standard

CM

HCWE/SE

KET

0 50 100 150 200

FID

/ m

VTime / s

Standard

CMTAG

FFAALC

ST

BA C

87

Figure 3.9 - (A) Representative FEG-SEM image of the PEG-AuNRs and (B) Histogram of length and (C)

width determined by measuring about 100 particles using the software ImageJ. The average

aspect ratio (length/width) calculated from the mean 47.4/13.37 was about 3.5.

Source: By the author.

The resulting PEG-AuNRs were subsequently fused with the CM--Lap derived

vesicles through mechanical extrusion.(16) For this, CM--Lap and PEG-AuNRs were

coextrused through a 100 nm polycarbonate membrane several times to guarantee the

formation of homogeneous CM--Lap shell and completely coated particles. The CM--

Lap/PEG-AuNRs ratio were varied to adjust the uniformity of the system and to avoid free

AuNRs. After fusion, the system was washed by centrifugation to remove an eventual excess

of CM--Lap and/or -Lap. The total concentration of -Lap in the final conjugates (OD750 nm

= 0.6) was around 2.5 µM. The low β-lap loading efficiency has also been observed in β-lap-

loaded micelles and polymeric particles.(128) Recently, Zheng et al proposed a system for

efficient loading and delivery of β-Lap using graphene oxides.(133)

A comparison of the UV-VIS-NIR spectra of PEG-AuNRs before and after the

conjugation is shown in Figure 3.10A. A slightly red shift is observed in presence of the

membrane and indicates the changing in the dielectric nature surrounding the nanorods.(154)

More pronounced, however, is the changing in the intensity of the longitudinal plasmonic

peak, which was significantly reduced. This effect has been attributed to the plasmon coupling

effect, which is typically induced by gold nanorods assembly.(155) This interpretation

matches with the FEG-SEM characterization (Figures 3.10E-F), which revealed that the

AuNRs are grouped inside the cell membrane vesicles. The average diameter of CM--Lap-

PEG-AuNRs, measured using the software Image J, was approximately 109 nm (Figure

3.11). FEG-SEM images of the CM-coated directly CTAB-AuNRs, i.e., without the

PEGylation of the AuNRs, show a totally different assembly (Figure 3.12) and suggest a

CTAB-mediated destabilization of cell membrane structure.

BA C

10 15 200

5

10

15

20

25

30

Fre

quency

Width / nm

30 40 50 60 700

5

10

15

20

25

30

Fre

quen

cy

Length / nm

88

The controlled assembly of the nanomaterials have been shown to be an interesting

way to fabricate systems with unique optical, electrical, and magnetic properties.(155-156)

This effect has been particularly interesting for sensing applications. For example, Wang et al

used controllable assemblies of AuNRs to detect an environmental toxin, called microcystin-

LR (MC-LR).(155) For this, side-by-side and end-to-end assemblies were fabricated and

when in presence of increasing concentrations of MC-LR, the assemblies were little by little

being disrupted. They found a relationship between the size of the assembly and the intensity

of the longitudinal peak with the concentration of MC-LR. Furthermore, although individual

AuNRs are already considered excellent surface enhanced raman scattering (SERS) platforms

with enhancement factors on the order of 104-10

5 due their anisotropic shape, their assemblies

have provided unique SERS enhancing properties.(157) The assembly creates coupled

plasmon modes, which results in regions of enhanced electromagnetic field, known as hot

spots, at the junctions.(157)

89

Figure 3.10 - Characterization of gold nanorods coated with cell membrane loaded -Lapachone (CM--Lap-

PEG-AUNRs) and comparison with PEG-AuNRs. (A) UV-VIS-NIR spectra and (B)

hydrodynamic diameter obtained from DLS measurements of PEG-AuNRs before and after

coating with CM--Lap. Longitudinal plasma resonance of PEG-AuNRs (λmax~750 nm)

decreased after the coating. C) Zeta potential of the AuNRs before and after functionalizations,

D) stability of CM--Lap-PEG-AuNRs in PBS buffer and 10% Fetal Bovine Serum; (E) and (F)

FEG-SEM images of CM--Lap-PEG-AuNRs showing the PEG-AUNRs grouped inside the cell

membrane vesicles.

Source: By the author.

1 10 100

5

10

15

20

25

Inte

nsity %

Hydrodynamic Diameter / nm

PEG-AuNRs

CM--Lap-PEG-AuNRs

300 450 600 750 900

Extinction / a

.u.

Wavelength / nm

PEG-AuNRs

CM--Lap-PEG-AuNRs

-30

-20

-10

0

10

20

30

Ze

ta P

ote

ntia

l /

mV

0 10 20 30 40 500

20

40

60

80

100

120 PBS buffer

10% FBS

Hyd

rod

yn

am

ic d

iam

ete

r / n

m

Time / hours

BA

DC

CTAB-

AuNRCM--Lap PEG-

AuNRCM--Lap-

PEG-AuNR

100 nm 100 nm

100 nm

FE

90

Figure 3.11 - (A) Representative FEG-SEM image of CM--PEG-AuNRs and (B) histogram determined by

measuring about 100 particles using the software ImageJ. The average diameter calculated was

about 109 nm.

Source: By the author.

Figure 3.12 - Representative FEG-SEM image of the CM--Lap-coated CTAB-AuNRs. No spherical cell

membrane vesicles are observed, suggesting their destabilization in presence of the surfactant

CTAB.

Source: By the author.

A comparative analysis of the hydrodynamic diameter of PEG-AuNRs with/without

CM--Lap by DLS measurements is shown in Figure 3.10B. The fixed angle of the DLS

instrument does not represent the true physical dimensions of the rod-shape particles, because

the hydrodynamic diameter can only be calculated by Stokes–Einstein equation for spherical

systems. However, the diffusion coefficient determined is still accurate and this technique can

be applied for a comparative analysis as demonstrated by Liu et al in the study of nanorod-

protein interactions.(158) Before the fusion, the PEG-AuNRs presented two different size

B

50 100 150 200 250 3000

5

10

15

20

25

30

Fre

que

ncy

Diameter / nm

200 nm

A

1 µm

91

distributions, which is consistent with their anisotropic shape, while the final CM--Lap-

PEG-AuNRs presented only one size distribution around 100 nm and agrees with the previous

results that show the assembly of the PEG-AuNRs inside the spherical vesicles.

The functionalization was further confirmed by zeta potential measurements (Figure

3.10C). The cell membrane presented a negative charge due mainly to the presence of

phospholipids and proteins. The zeta potential of AuNRs changed from highly positive, due

the CTAB stabilization, to close to zero after PEGylation. The surface charge of the PEG-

AuNRs upon fusion with CM--Lap was about the same of the CM--Lap vesicles,

suggesting successful coating.

To examine the serum stability of the resulting CM--Lap-PEG-AuNRs, they were

resuspended in 1x PBS and 100% FBS solution and the hydrodynamic diameter was

monitored by DLS. All samples were incubated at 37ºC and gentle shacked prior the

measurements (Figure 3.10D). No major change was observed, indicating that the CM--

Lap-PEG-AuNRs present good stability either in buffer or biological media. The ability of

CM--Lap-PEG-AuNRs to avoid the binding of proteins from the biological media was

evaluated by Isothermal titration calorimetry (ITC) (Figure 3.13). The interactions between

bovine serum albumin (BSA), a major representative globular protein present in the serum,

and PEG-AuNRs, CM--Lap-PEG-AuNRs, and CTAB/PEG-AuNRs were evaluated.

CTAB/PEG-AuNRs consist in the PEGylation of the CTAB-AuNRs using a lower

concentration of mPEG-SH, i.e., just enough to keep the particles stable in 0.1x PBS,

resulting in a zeta potential of +18 mV. In the tested conditions, no interaction was observed

for PEG-AuNRs and CM--Lap-PEG-AuNRs, while the binding between BSA and

CTAB/PEG-AuNRs is an exothermic process. It is expected that the PEGylation of

nanoparticles would reduce protein adsorption on the nanoparticle surface as a result of steric

repulsions and hydrophilization induced by PEG chains, and previous studies have also shown

no heat exchange when BSA is added to PEGylated nanoparticles.(148, 159) Since the CM--

Lap-PEG-AuNRs presented a similar behavior, we can infer that they also have some

properties characteristic from PEGylated particles such as stability in physiological media and

ability to avoid the BSA binding. These results suggested that the coating of the nanoparticles

with cancer cell membrane vesicles may avoid the opsonization. However, further and more

detailed studies need to be performed to prove this hypothesis.

92

Figure 3.13 - Plots of heat flow vs time of microcalorimetric titration of (a) PEG-AuNRs; (b) CM--Lap-PEG-

AuNRs and (c) CTAB/PEG-AuNRs by BSA solution. Injections of 2 μL, at 25 °C, of 0.01 mM of

BSA solution were made on nanoparticles suspensions. Control of BSA dilution was carried out

by injections of BSA solution in buffer.

Source: By the author.

Together, these results demonstrate that the coating was efficient and the gold

nanorods are grouped inside the cell membrane vesicles. According to previous studies,(17-

18) the coating of nanoparticles with natural cell membrane occurs with the accurate

transference of the membrane bilayer structure onto the particle surface, leading to a right-

side-out conformation. This is important to maintain the surfaces antigenic diversity and

functions of source cells, besides the increased stability in biological media and longer

circulation time. Furthermore, the cell membrane vesicles prepared by this approach present

some advantages with respect to the exosomes, such as versatility to be made in different

sizes and loaded with many types of therapeutic agents, and reproducibility, all clinically

relevant characteristics.(20) Also, exosomes may lead to more safety concerns due to their

more complex content, such as miRNA.

3.3.3 Photothermal properties

Photothermal therapy has been increasingly investigated as a minimally invasive

alternative for cancer treatment.(7) Having verified the formation of the CM--Lap-PEG-

AuNRs conjugates, we investigated their photothermal properties using a 808 nm laser at a

power density of about 1.5 W/cm2, which is consistent with earlier experimental conditions

used in PPTT to guarantee minimal side effects,(55,105,160) and an optical thermometer to

monitor the temperature. The 808 nm wavelength overlaps with the longitudinal absorption

band of the AuNRs and it is important due to the low absorption of the tissue in the NIR.(8)

Equal optical densities (OD) have been shown to be an appropriated method to

compare the photothermal properties of nanoparticles.(10) Figure 3.14A shows that as the

suspensions of CM--Lap-PEG-AuNRs and PEG-AuNRs, both with OD750 nm of about 0.6,

0 10 20 30 40 50 60

-0.3

-0.2

-0.1

0.0

Time / min

µca

l /

sec

0 10 20 30 40 50 60

-0.3

-0.2

-0.1

0.0

Time / min

µca

l /

sec

0 10 20 30 40 50 60

-0.3

-0.2

-0.1

0.0

Time / min

µca

l /

sec

BA C

93

were exposed to NIR laser, the temperature in both systems increased. Closer examination

revealed some slight differences mainly in the initial period (inset of Figure 3.14A). First, the

curve slope in the first minutes after laser was turned on was greater for PEG-AuNRs than to

CM--Lap-PEG-AuNRs, but after about 2.5 min, they seem to be almost the same. This could

be ascribed to the assembly of nanorods observed on CM--Lap-PEG-AuNRs system.

Initially, the AuNRs are grouped inside the CM vesicles, which decrease their absorption at

NIR region (Figure 3.10A). The temperature increases generated by the laser irradiation

would destabilize the cell membrane structure and release both PEG-AuNRs and -Lap, and

as they are being released, their photothermal behavior is becoming more likely the free PEG-

AuNRs. Besides the assembly of the gold nanorods, the cell membrane layer by itself

contributed to the slower temperature rise of CM--Lap-PEG-AuNRs compared to the their

PEGylated counterparts, i.e., the additional biological layer may hamper the heat dissipation

into the surrounding media.(161) The CM--Lap-PEG-AuNRs also take more time to

stabilize, which suggest that some changes in their structure still occur even after 10-15 min

of irradiation. No significant temperature change was observed when buffer solution (data not

shown) or cell culture media were irradiated with the NIR laser at the same conditions.

The FEG-SEM images before and after 10 and 25 min of laser irradiation shown in

Figure 3.14D-F, respectively, clearly show the gradual release of the PEG-AuNRs from the

CM vesicles with the laser irradiation time. It is worth stressing that the laser irradiation did

not cause any shape modification, i.e., in size and morphology of the AuNRs. This is

important since a laser irradiation sufficient to modify the structure of the nanoparticles is

probably able to induce cell dead and side effects. Moreover, it has been shown that the

smaller nanoparticles resulting from this reshaping process present high cytotoxicity.(162)

94

Figure 3.14 – (A) Representative of temperature change vs time in suspensions (optical density at 750 nm = 1)

irradiated with 808 nm laser with power density of 1.5 W/cm2, (B) Increase of the longitudinal

band with the laser irradiation, (C) release of -Lapachone at different time of irradiation. SEM

images of CM--Lap-PEG-AuNRs (D) before, (E) after 10 min and (F) after 20 min of laser

irradiation. The laser irradiation increases the temperature of the suspension, breaks the vesicles

and causes the release of the AuNRs.

Source: By the author.

The increasing in the longitudinal band of the AuNRs with the increasing of the laser

irradiation (Figure 3.14B) is expected and also revealed the disruption of the cell membrane

vesicles and release of the PEG-AuNRs. Taken together, these results show that CM--Lap-

PEG-AuNRs present a slower temperature rise compared to their PEG-AuNRs, but the

disruption of the membrane occurs in the first minutes of NIR irradiation, and their behaviors

become equals.

The release of -Lap was analyzed by dividing the sample in four aliquots, irradiating

them with the laser for different times, centrifuging them immediately to separate the released

-Lap from the nanoparticles using centrifugal filters, and measuring the typically absorbance

of the -Lap at 257 nm to calculate the correspondent concentrations. The calculated released

concentrations with the laser irradiation time are shown in Figure 3.14C. As expected, the

amount of -Lap released increases with the irradiation time, which is in good accordance

with previous studies.(55,163) Accordingly, laser-induced release of molecules on surface of

gold nanorods is a thermal process and the amount of released molecules is dependent upon

the laser power and period of irradiation.(55,163) However the total concentration of -Lap

0 5 10 15

0

10

20

30

Cell culture media

PEG-AUNRs

CM--Lap-PEG-AuNRs

T

/

C

Time / min

Laser

ON

1.8 2.7 3.6

5

10

15

BA

400 600 800

Extinction / a

.u.

Wavelength / nm

0 min

10 min

20 min

30 min

ED F

C

5 10 15 20

0.6

0.9

1.2

Concentr

ation /

M

Time / min

95

released from CM--Lap-PEG-AuNRs is very low (< 1.2 µM) even after 20 min of

irradiation.

3.3.4 In vitro photothermal therapy

For the in vitro photothermal therapy experiments we have used the same optical

density for both samples with/without -Lap (final OD750 nm in cell culture media = 0.3),

which allows quantitative comparison of the photothermal properties for all systems.(55) The

cells were incubated for 4 hours with the particles, washed twice with PBS, and left in cell

culture media. After 10 min of irradiation, the cells were incubated for 2 h at 37ºC and 5%

CO2, followed by staining with trypan blue to evaluate the viability (Figure 3.15, 3.16).

Surprisingly, an increasing number of dead cells stained by trypan blue are observed for cells

treated with CM--Lap-PEG-AuNRs compared to the cells treated with CM-PEG-AuNRs.

Since the only difference between these two systems is the presence of -Lap, we can infer

that even at low concentrations (probably below 1 µM), the -Lap is contributing for the

toxicity of the CM--Lap-PEG-AuNRs and apparently it has been released only after laser

irradiation. It is noteworthy that only the cells in the laser spots (highlight areas) were

damage.

Figure 3.155 - Photothermal therapy of A549 cancer cells treated with CM--Lap-PEG-AuNRs and CM-PEG-

AuNRs. The highlight areas represent approximately the laser spots (~1.5 W/cm2, 10 min) on the

samples. Dead cells were stained with Trypan Blue. Cells without the particles are not affected by

the laser irradiation while cells incubated with CM--Lap-PEG-AuNRs and CM-PEG-AuNRs

were visibly injured.

Source: By the author.

Laser

Control CM-PEG-AuNRsCM-Lap-PEG-AuNRs

No laser

96

Figure 3.16 - Photothermal therapy of A549 cancer cells treated with CM--Lap-PEG-AuNRs and CM-PEG-

AuNRs for 4 h. The highlight areas represent approximately the laser spots (~1.5 W/cm2, 10 min)

on the samples. Dead cells were stained with Trypan Blue. Cells without the particles are not

affected by the laser irradiation while cells incubated with CM--Lap-PEG-AuNRs and CM-PEG-

AuNRs were visibly injured.

Source: By the author.

It is known that the cytotoxicity effect of -Lap is depending on NAD(P)H:quinone

oxidoreductase (NQO1), which is overexpressing in several cancers, including A549 lung

cancer cell line.(124) Park et al have been shown that heating the cells at mild temperatures

(41-42ºC) elevates NQO1 expression in various cancer cells, including A549, increasing the

anticancer effects of -Lap.(127) Accordingly, heating the cancer cells to 42ºC increased the

NQO1 level for 1 h and remained elevated for 72 h. This might explain the significant effect

of -Lap in the cytotoxicity of A549 cells, even at very low concentrations. Also, it highlights

that more than only two combined effects (photo and chemotherapy), the CM--Lap-PEG-

AuNRs can act as a synergistic system: the temperature increases resulted from the plasmonic

properties of AuNRs may enhance the anticancer properties of the -Lap.

The delivery of chemotherapeutic drugs and photothermal agents has been shown to

be an effective therapeutic approach for combined cancer treatment.(104, 164) The results

presented in Figures 3.15 and 3.16 collectively demonstrated the effectiveness of CM--Lap-

PEG-AuNRs as a multifunctional agent for combined chemo and photothermal destruction,

achieving synergistic cancer treatment.

Laser

No laser

Control CM-PEG-AuNRsCM-Lap-PEG-AuNRs

97

3.3.5 Cellular uptake

After investigation of the in vitro photothermal properties, the ability of the CM--

Lap-PEG-AuNRs to target and uptake A549 cancer cells were analyzed. First, the uptake was

investigated by fluorescence confocal analysis. CM were stained with CellMaskTM

plasma

membrane stain (Invitrogen), which consists in amphipatic molecules, and then conjugated

with the PEG-AuNRs as described previously. For this specific confocal experiment, CM

were not loaded with -Lap, since the goal was observe only the internalization ability and the

-Lap could interfere in the label with the fluorophore. The confocal image of A549 cells

incubated with CM--Lap-PEG-AuNRs for 4 hours (Figure 3.17A) shows that the particles

were internalized and localized in the cell cytoplasm. The discrete red spots in the cytoplasm

may be an indicative of endosomal uptake of the CM-PEG-AuNRs inside de cells.(46) In

Figure 3.18, the strong signal observed for CM-PEG-AuNRs when nucleus is in focus

(middle rows) confirms the cellular uptake. These observations corroborate a previous study

in which MDA-MB-435 cell membrane-coated PLGA nanoparticles were incubated with

MDA-MB-435 cells.(18)

Since not much information is available about CellMask stain and to elucidate whether

the excess of the fluorophore was removed or whether CellMask could be release from the

CM vesicles in the presence of the cells, we performed the same experiment using only the

CellMask stain as a control. The cells were treated with high concentrations of CellMask and

incubated for 4 hours. Confocal images comparing both systems are shown in Figure 3.20,

and revealed differences between them. In the 3D images (Figures 3.20C, 3.20F), these

differences are even more evident, showing that in the A549 cells incubated with only

CellMask stain the red dots are in the cell membrane and not in the cytoplasm like CM--

Lap-PEG-AuNRs.

Since the plasma membrane stain could alter the surface of the CM and, consequently,

the uptake, the internalization of CM--Lap-PEG-AuNRs was further evaluated by UV-Vis-

NIR spectroscopy. This simple method has been shown to be very useful to investigate the

uptake of plasmonic nanostructures and, according to previous studies, the concentrations of

nanoparticles determined is consistent with those obtained by inductively coupled plasma

mass spectrometry (ICP-MS).(149) The cells were incubated with PEG-AuNRs and CM--

Lap-PEG-AuNRs with a final OD at 750 nm of 0.3. Based on the optical properties of both

systems shown in Figure 3.10A, we probably have used a higher concentration of AuNRs at

98

CM--Lap-PEG-AuNRs compared to PEG-AuNRs, and therefore this method was used only

as a comparative and qualitative analysis. At each point time, cells were lysed in hypotonic

buffer, followed by sonication to break big molecular aggregates. The absorption was

normalized by the number of cells at each point. Figure 3.17B shows time dependence of the

extinction at 750 nm of the cells treated with CM--Lap-PEG-AuNRs, i.e., extinction at 750

nm increases with the incubation time. In Figure 3.17C, which represents the extinction

spectra of the cells incubated with PEG-AuNRs and CM--Lap-PEG-AuNRs for 4 h

corrected by the number of cells and normalized, the extinction of CM--Lap-PEG-AuNRs

was obviously superior compared to PEG-AuNRs.

Figure 3.17 - (A) Confocal images of A549 cells after cell exposure to CM-PEG-AuNRs for 4 h. Blue: cell

nuclei (Hoechst 33258). red: CellMask Deep Red-labeled CM-PEG-AuNRs (Exc/Em = 649/667

nm). (B) Time dependence of the extinction at 750 nm of the cells treated with CM--Lap-PEG-

AuNRs, showing the dynamic of the uptake of the particles. The data were corrected by the

number of cells (relative ext) and are showed as mean ± SD of triplicate. (C) Comparison of the

uptake of PEG-AuNRs and CM--Lap-PEG-AuNRs after 4 h of incubation. The extinctions

were corrected by the number of cells and normalized.

Source: By the author.

700 8000.0

0.5

1.0

Norm

aliz

ed e

xt

/ a

.u.

Wavelength / nm

PEG-AuNRs

CM--Lap- PEG-AuNRs

20 µm

BA C

0 2 4 6 8 10 12

0.100

0.104

0.108

Re

lative

ext

=

75

0 n

m /

a.u

.

Time / h

99

Figure 3.18 - Intracellular uptake of CM-PEG-AuNRs. Samples were incubated with A549 cells for 4 h. Red:

CellMask Deep Red-labeled CM-PEG-AuNRs (Exc/Em = 649/667 nm); Blue: nuclei stained

with Hoechst 33258. CM-PEG-AuNRs show strong signal when nucleus is in focus (middle

row), demonstrating cellular uptake.

Source: By the author.

100

Figure 3.1916 - Confocal images of A549 cells after cell exposure to CellMask Deep Red stain (a, b, c) and

CellMask-stained CM-PEG-AuNRs (d, e, f) for 4 h. Blue: cell nuclei (Hoechst 33258).

Source: By the author.

Together, these analyses provide evidence that the functionalization with CM

facilitates the uptake in the source cells. This observation is in agreement with previous

studies in which it is shown that MDA-MB-435 cell membrane-coated PLGA polymeric

nanoparticles presents a significantly higher uptake in MDA-MB-435 cells compared to both

bare PLGA nanoparticles and red blood cell membrane-coated PLGA nanoparticles.(18)

According to the authors, the coating of nanomaterials with cancer cell membrane can

preferentially increase their affinity to the source cancer cells and they attributed this effect to

the transference of cell adhesion molecules with homotypic binding properties.(18) Since

many cancer cells express surface antigens with hemophilic adhesion domains,(165) which

are responsible for multicellular aggregate formation in tumors, we believe that this effect

could also be responsible for the increasing uptake of CM--Lap-PEG-AuNRs compared to

PEG-AuNRs. Furthermore, the applications of this new system could be optimized and

extended for other applications, such as radiotherapy, since -Lap has shown to be a very

promising anticancer agent for radiosensitization.(134, 166-167)

A

B

C

D

E

F

101

3.3.6 Cytotoxicity

To evaluate the safety of the CM--Lap-PEG-AuNRs, their cytotoxicity in absence of

laser irradiation was investigated using two colorimetric assays: crystal violet (CV) and MTT.

Equivalent doses of PEG-AuNRs, CTAB-AuNRs, CM and CM--Lap were tested in parallel

for comparison. The goal was to analyze the behavior of the CM--Lap-PEG-AuNRs against

their source cell (A549), another type of cancer cell (HTC), and a healthy one (HepaRG). The

results are shown on Figure 3.20A-B and reveal a good agreement between both assays. No

significant cytotoxicity for any of tested samples was observed, except for CTAB-AuNRs.

The cationic surfactant CTAB has been shown to be very toxic to cells at very low

concentrations.(168) The complete removal of CTAB from AuNRs and their functionalization

with other molecules is a crucial step to guarantee stability and biocompatibility for medical

applications. The results proved that PEGylation and CM coating of the AuNRs were efficient

to prevent cytotoxivity at the tested concentrations.

In agreement with data in the literature,(124, 128, 130) the specific toxicity of -Lap

for cancer cells was found (Figure 3.20C). -Lap induced cell death of A549 and HTC cells

at a concentration of 4 and 8 µM, while HepaRG showed to be more resistant. For A549 cells,

it is possible to observe that while a dose of 8 µM causes a cell death of more than 50 %, 4

µM or 2 µM has almost no effect. These sharp dose-responses of -Lap have been

reported.(169) It is worth to emphasize that neither CM--Lap nor CM--Lap-PEG-AuNRs

present any significant toxicity, which indicates that the concentration of -Lap released from

these structures in absence of laser irradiation is very low.

102

Figure 3.170 - Cell viability, evaluated by (a) MTT; (b) Crystal Violet, and (c) MTT of A549, HTC and

HepaRG cells after incubation with the samples for 24 h. In (a) and (b) the columns blue, green,

and red represent the quantities of 5, 10 and 15 µL, respectively. In (c) the columns red, green,

blue, light blue and pink represents the concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 µM of -Lap,

respectively. The relative percentage of viable cells was calculated by considering the control as

100% of viable cells. The results represent the mean ± SD of data normalized to untreated controls

from two independent experiments in triplicate. *P<0.05 with respect to untreated controls.

Source: By the author.

3.3.7 Detection of Intracellular Reactive Oxygen Species (ROS)

One of the mechanisms of -Lap toxicity proposed in literature is through the

generation of ROS. NQO1 metabolizes -Lap to a very reactive hydroquinone, which is

oxidized back to semiquinone or quinine in a futile cycle metabolism.(169) In this process,

semiquinones, free radical generators, initiate the redox cycle, and ROS including superoxide

and hydrogen peroxide are then generated.(170) The effect of -Lap after conjugation with

CM--Lap or CM--Lap-PEG-AuNRs was evaluated by 2',7'-dichlorodihydrofluorescein

Control

CTAB-AuNR

PEG-AuNR CM

CM-b-Lap

CM-PEG-A

uNR

CM-b-Lap-PEG-A

uNR0

20

40

60

80

100

**

% C

ell

via

bili

ty

*

Control

CTAB-AuNR

PEG-AuNR CM

CM-b-Lap

CM-PEG-A

uNR

CM-b-Lap-PEG-A

uNR0

20

40

60

80

100

% C

ell

via

bili

ty

***

A549

HTC

HepaRG

Control

CTAB-AuNR

PEG-AuNR CM

CM-b-Lap

CM-PEG-A

uNR

CM-b-Lap-PEG-A

uNR0

20

40

60

80

100

*

*

% C

ell

via

bili

ty

*

A549

Control

CTAB-AuNR

PEG-AuNR CM

CM-b-Lap

CM-PEG-A

uNR

CM-b-Lap-PEG-A

uNR0

20

40

60

80

100

**

% C

ell

via

bili

ty

*

HTC

Control

CTAB-AuNR

PEG-AuNR CM

CM-b-Lap

CM-PEG-A

uNR

CM-b-Lap-PEG-A

uNR0

20

40

60

80

100

*

*

% C

ell

via

bili

ty

*

HepaRG

Control

CTAB-AuNR

PEG-AuNR CM

CM-b-Lap

CM-PEG-A

uNR

CM-b-Lap-PEG-A

uNR0

20

40

60

80

100

% C

ell

via

bili

ty

*

**

Control

b-Lap 0

20

40

60

80

100

% C

ell

Via

bili

ty

*

*

A549

Control

b-Lap 0

20

40

60

80

100

*

% C

ell

Via

bili

ty

HTC

Control

b-Lap0

20

40

60

80

100%

Ce

ll V

iab

ility

HepaRG

A B C

103

diacetate (H2DCFDA) and compared with free -Lap (Figure 3.21). The data reveal that -

Lap increased the intracellular ROS generation only in A549 and HTC cells, but not in

HepaRG cells, and no significant ROS was generated by CM--Lap or CM--Lap-PEG-

AuNRs, which is in good accordance with the cytotoxicity results.

Figure 3.21 - Measurement of generation of ROS in A549 and HepaRG cells. (a) Cells were incubated with the

samples for 2 hours and the generation of ROS was measured using the 2',7'-

dichlorodihydrofluorescein diacetate (H2DCFDA) assay. The relative percentage was calculated

by considering the control as 100%. Data are expressed as mean ± SD of triplicate from two

independent experiments. *P<0.05 with respect to untreated controls.

Source: By the author.

The effect of the ROS generation by β-lap has been widely investigated. Park et al

suggest that upregulation of intracellular ROS is involved in -Lap induced programmed

necrosis, called necroptosis.(170) Early study demonstrated by comet assay that accumulation

of ROS such as hydroxyl radicals generated by β-lap-loaded PEG-PLA as well as free β-lap

causes severe DNA damage.(128)

3.4 CONCLUSIONS

We have demonstrated the successful coating of PEG-AuNRs into cellular membranes

vesicles purified from A549 cancer cells and loaded with the anticancer agent -Lapachone.

The characterization of the system demonstrated the assembly of PEG-AuNRs inside the

vesicles and NIR irradiation led to disruption of the vesicles and release of the PEG-AuNRs

and -Lapachone. In vitro studies also revealed that this multifunctional noncovalent platform

was capable to deliver therapeutic molecules and plasmonic nanostructures upon NIR

A549 HepaRG

Control

PEG-AuNR CM

CM-b-Lap

CM-PEG-AuNR

CM-b-Lap-PEG-A

uNRb-L

ap0

50

100

150

200

250

300

350

RO

S g

en

era

tio

n

(%

co

ntr

ol)

Control

PEG-AuNR CM

CM-b-Lap

CM-PEG-AuNR

CM-b-Lap-PEG-A

uNRb-L

ap0

50

100

150

200

250

300

350

*

*

RO

S g

en

era

tio

n

(%

of co

ntr

ol)

104

irradiation, resulting in an enhanced cytotoxicity against A549 cancer cells. The NQO1-

specific cytotoxicity of -Lap combined with heat generated by laser irradiation of the AuNRs

result in a synergic toxicity specific for cancer cells. No cytotoxicity was observed in absence

of laser irradiation. Further studies would be focus on the optimization of the loading of -

Lap and a detailed investigation of their in vitro and in vivo photothermal cytotoxicity

mechanism.

The system we describe differ from others that already exists mainly because of the

toxicity activated by NIR radiation and the synergic effect demonstrated between the heat

generated by laser irradiation and -Lapachone activity. Its major potential advantages

consists in its specific toxicity against cancer cells added to its capability to avoid

opsonization and target specific cancer sites, versatility and multifunctionality, allowing the

fabrication with different plasmonic systems. The system can mimic the cancer cells

properties and represents new opportunities for the development of more efficient methods for

diagnosis and treatment of cancer, increasing the chances of a successful therapy with

reducing side effects. Furthermore, this multifunctional platform for activated drug release

and phototherapy is a promised candidate for applications in personalized medicine. Other

potential utilizations may include radiotherapy, photodynamic therapy, and two photon

luminescence imaging.

105

4 Gd(III)-ENCAPSULATING Au NANOMATRYOSKHAS: ENHANCED

T1 MAGNETIC RESONANCE CONTRAST IN A THERANOSTIC

NANOPARTICLE

4.1 INTRODUCTION

4.1.1 Magnetic Resonance Imaging (MRI)

Magnetic resonance imaging (MRI) is currently the most used biomedical imaging

modality.(21) It is a non-invasive technique with contrast versatility and high spatial and

temporal resolution.(22) Unlike X-Ray Computer Tomography, Single-Photon Emission

Computed Tomography or Positron Emission Tomography, no harmful high-energy radiation

is used in MRI.(171)

MRI derives directly from nuclear magnetic resonance (NMR), a widely used

technique to determine molecular structure. MRI focuses in one type of atomic nucleus, the

hydrogen in H2O, termed “H proton”.(21) The 1H proton can be considered as a charged

sphere rotating with a magnetic moment and collinear angular magnetic momentum.(21)

Under an external magnetic field (B0), each proton rotates around its axis with an angular

frequency ω0, which is proportional to the applied magnetic field strength according to the

Larmor equation: ω0 = γB0 (γ is the gyromagnetic ratio). This produce a net magnetization in

the same direction of B0 (M), which is described by its components: Mz, parallel to B0 and

called as longitudinal magnetization, and Mxy, perpendicular to B0 and known as transverse

magnetization. When radiofrequency (RF) energy is applied in “resonance” with the ω0 of the

protons, the net magnetization can be flipped away from its original axis, a process called

excitation.(21, 172) Return to equilibrium results in emission of MR signal proportional to the

number of excited protons in the sample. The return of the net magnetization to its original

equilibrium state (Mz(0)) is known as longitudinal (T1) relaxation or spin–lattice relaxation

(Figure 4.1), and consists in the loss of the energy from the excited state (spin) to it

surrounding tissue (lattice). The recovery of net longitudinal magnetization is governed

by:(172)

(

⁄ ) (4.1)

106

Where T1, called longitudinal relaxation time, is defined as the time required for protons to

return to 63% of their longitudinal magnetization, and it is reciprocal (1/T1) is known as T1

relaxation rate.(172) Protons from different localizations in biological samples, such as

mobile water, tissue-bond water, have different T1 relaxation rates.(172) Therefore, when a T1

–weighted MRI image is acquired, tissues containing different types of protons produce

images of different signal intensity, and consequently, with different contrast.

The loss of the magnetization in the xy plane is known as T2 relaxation, or transverse

relaxation, and it is an exponential decay due the spin-spin interactions of precessing nuclei

resulting in the loss of phase coherence. In this case, upon excitation of protons by an RF

pulse, the 90º RF pulse produces phase coherence of the individual protons and generates the

maximum Mxy,(172) As the recovery in longitudinal magnetization takes place, the transverse

magnetization begins to decrease (T2 relaxation), called decay (Figure 4.1). T2 relaxation time

is defined as the time it takes for the transverse magnetization to decay to 37% of its original

value:

⁄

(4.2)

In general, T2 relaxation is faster than that of T1. Samples in which protons have a

higher degree of translational and rotational freedoms, for example mobile waters compared

to tissue bound waters, generally have longer T2 values, which allows to link T2 with specific

properties of the tissue and it is also very useful in medicine.(172)

107

Figure 4.1 - Schematic showing the main differences between T1 and T2 relaxation.

Source: Adapted from KHEMTONG et al.(172)

For many MRI applications, a substance is required to induce additional contrast in

regions of interest and produce more precise diagnosis, called contrast agents. They are

paramagnetic, superparamagnetic, and ferromagnetic compounds that catalytically shorten the

relaxation time of water protons.(173) These agents shorten both T1 and T2, but they are

usually classified as T1 or T2 contrast agent based on whether the substance increases in a

greater extent the longitudinal or transverse relaxation, respectively. Therefore, T2-weighted

contrast agents locally modify the spin-spin relaxation process of water protons, producing

negative or dark images. T2 contrast agents are based on materials such as superparamagnetic

Fe3O4, MnFe2O3, FeCo, and FePt nanoparticles. T1-weighted contrast agents affect nearby

protons through spin-lattice relaxation, producing positive or bright image contrast. They are

based on paramagnetic materials such as Gd(III) and Mn(II)).(23) The ability of a contrast

agent to change the longitudinal (1/T1) or transverse (1/T2) relaxation rate is represented as r1

or r2, respectively.(173) Relaxivity is defined as the change in relaxation rate after the

introduction of the contrast agent normalized to the concentration of the contrast agent

[M]:(173)

(

⁄ )

(4.3)

(

⁄ )

(4.4)

m

wo

Bo

Bo

wom

Loss of magnetization in the z

plane due to spin-lattice

interaction (slow)

Loss of magnetization in the xy

plane due to spin-spin

interaction (fast)

dark

bright

108

In T1 agents, a chemical exchange between bound and free water molecules is required

for relaxation process, while T2 contrast produce much stronger magnetic susceptibility,

affecting a large number of water molecules.(172) Despite their utility, T2 contrast agents

have several disadvantages that limit their use in clinical applications. They cause a reduction

in the MRI signal (dark images), which can be confused with other pathogenic conditions

(such as blood clots and endogenous iron). In the case of tumor imaging, they can also induce

magnetic field perturbations on the protons in neighboring normal tissues, which can make

spatially well-resolved diagnosis difficult.(24) In contrast, T1 contrast agents increase the

specificity and sensitivity of the MR image by producing bright images. Among the

paramagnetic materials useful for T1 contrast MR imaging, Gd(III) is the most effective

contrast agent currently available for clinical use.

4.1.2 Gd(III) based contrast agents

The overwhelming majority of contrast enhanced MRI clinical exams are performed

with Gadolinium complexes.(173) Gd(III) possesses seven unpaired electrons, and the spin of

which perturbs the proton relaxation in water results in an efficient shortening of longitudinal

relaxation time and increases the magnetic signal intensity.(174) However, aqueous

gadolinium ions present high toxicity in its free state, and chelation by a co-ligant is

required.(175) Gd(III)-diethylenetriamine pentaacetic acid (DTPA) (Magnetivist®) is among

the most popular clinical agent. It has been used to enhance tissue pathology, detect leaks in

the blood-brain barriers (BBB), and in some cases identify physiological changes in

tissue.(176) Table 4.1 shows the r1 relaxivity for six clinically used Gd(III) chelates at 0.41 T

and 1.41 T.

Table 4.1 - T1 relaxivity for clinically used contrast agents at 0.47 and 1.41 T.

Complex r1 (mM

-1 s

-1)

B0 = 0.47 T

r1 (mM-1

s-1

)

B0 = 1.41 T

Gd-DTPA (Magnevist®) 3.8 3.4

Gd-DOTA (Dotarem®) 3.5 3.1

Gd-DTPA-BMA (Omniscan®) 3.8 3.6

Gd-HP-DO3A (ProHamce®) 3.6 3.2

Gd-BOPTA (MultiHance®) 4.8 4.1

Gd-DO3A-butrol (Gadovist®) 3.7 3.2

Source: Adapted from LAURENT et al.(177)

109

Recently several studies have raised serious concerns regarding the safety of Gd(III)

chelates.(178-180) For example, nephrogenic system fibrosis (NSF), a progressive and

potentially fatal condition, is associated with gadolinium chelates and occurs mainly in

patients with decreased renal function.(178,180) These side effects are aggravated due the low

relaxivity of Gd(III) chelates, which makes them effective only at high concentrations (>0.1

mM),(173) as well as their instability and the release of small amount of free Gd(III)

ions.(174) Furthermore, they are based on small molecules and present some additional

disadvantages such as short blood circulation time and rapid extravasation from the vascular

space,(181) limiting their applications, especially for cancer imaging. To overcome these

challenges, recent efforts have been focus in increasing the sensitivity and safety of Gd(III)-

based contrast agents for MRI. High relaxivity contrast agents can be used at lower doses, or

can be used in molecular imaging to detect low concentration targets.(182)

4.1.3 Nanostructured T1 MRI contrast agents

Nanomaterials provide excellent platforms for the development of MRI contrast

agents.(183) Many nanostructured agents have been developed by loading Gd(III) chelates

into polymeric nanoparticles,(184) dendrimers,(185) micelles,(186) lipossomes,(175) and

mesoporous silica nanoparticles.(187) Moreover, Gd-chelates have been conjugated on the

surface of gold nanospheres (188-189) or even with gold nanoparticles.(190) The

incorporation of Gd(III) onto or into nanoparticles enhance their sensitivity by increasing the

concentration of Gd(III) per nanocarrier, prolonging imaging time, and reducing their

toxicity.(22, 28-32) Moreover, these systems usually present higher relaxivity compared to

free Gd(III)-chelates due both an addictive effect of all Gd(III) centers, and the slow global

rotational motion that enhances the r1.(191) An overview of different types of nanostructured

T1 MRI contrast agents and their respective relaxivity r1 are shown in Table 4.2.

Many are the factors that influence the relaxivity of nanostructured T1 agents. As can

be seen from Table 4.2, size and shape play a significant role in the r1 relaxivity. The position

of the Gd(III) chelate also influences the relaxivity. For example, if the chelates are on the

surface of the particle, it will be more exposed to the bulk water molecules which eventually

results in an enhanced T1 relaxation effect.(192) Another important factor is the applied

magnetic field. Higher magnetic fields, such as 4.7 T, are relevant because they provide not

110

only a greater signal to noise ratio, but also a higher spatial resolution and reduced acquisition

times.(182) Many MRI scanners with higher fields, such as 3, 4.7 or even 9.4 T, have been

used clinically. However, T1 relaxivity of Gd(III) compounds typically decreases as the

magnetic field increases.(182, 193) Caravan et al. performed a detailed study to understand

the dependence of r1 and r2 relaxivity as a function of magnetic field using both experimental

and theory.(182) They show that the effect of the magnetic field in the relaxivity is more

pronounced for slow tumbling molecules than for fast tumbling molecules. For example,

small molecules like Gd-DTPA show a modest decrease in r1 with field, however, for Gd(III)

chelates bound to serum albumin, the relaxivity decrease from 24.3 mM-1

s-1

at 1.4 T to 11.2

mM-1

s-1

at 4.7 T.

Compared to traditional small molecular-based contrast agents or therapeutic drugs,

these new nanomedicine platforms permit a highly integrated design that can incorporates

multiple functions such as imaging, targeting, and therapeutic in one system.(172) These

modular systems with theranostic functions have been proved to be essential for overcoming

the challenge of tumor heterogeneity and achieving personalized medicine. Moreover, the

precise localization of nanomaterials in the body is one important step to evaluate the

efficiency and safety of these new therapeutic agents.(115) In this context, Gd(III)-chelates

conjugated with near infrared (NIR) phothermal transducers have attracted much attention.

Coughlin et al conjugated Gd(III) chelates on the surface of gold nanoshells.(194) The system

presented a MRI contrast enhancement efficacy (37 mM-1

s-1

at 1.41 T, 37ºC) and higher

signal intensity in vivo after an intratumoral injection in a subcutaneous melanoma tumor.

This enhancement was attributed to the high payload of Gd(III) ions and the restricted

molecular tumbling of the Gd(III)-chelates when conjugated with the nanoshells. Rotz et al

conjugated Gd(III)-chelates to a DNA-functionalized gold nanostars with good high relaxivity

of r1 of 54.7 mM-1

s-1

at 1.41 T and 9.4 mM-1

s-1

at 7 T.(195) Besides the high payload of

Gd(III) ions and the restricted molecular tumbling of the Gd(III)-chelates, the authors

attributed the enhancement of the relaxivity to the surface curvature on the nanostar surface.

According to them, the shape affect the organization of conjugated DNAs on the particle

surface and, consequently, the sequestration of water molecules in the proximity of the

Gd(III) complexes.(195) However, these systems can face many limitations for medical

applications. First of all, in the nanoshells case, the size of the particles can difficult their

accumulation after an intravenous injection for treatment of other kinds of tumor. Moreover,

negatively or positively charged nanomaterials can led to a rapid formation of protein corona

when in presence of biological fluids,(196) which can change their pharmokinetcs and

111

physico-chemical properties. Finally, the fact that the Gd(III) chelates are in the surface of the

nanomaterial can lead to the same safety concerns related to the small molecules Gd(III)

agents.

Table 4.2 - T1 relaxivity of Gd(III) conjugated nanomaterials.

Complex r1 (mM-1

s-1

) Reference

Gd(III)-conjugated gold ganoparticles (5 nm) 50.0 (1.41 T)

11 (4.7 T)

(190)

Gd(III)-conjugated gold nanoshells 37.0 (1.41 T)

(31)

Gd(III)-conjugated gold nanostar 54.7 (1.41 T)

9.4 (7.0 T) (32)

Gold nanoparticles@DNA-Gd(III) 30 nm 20.0 (1.41 T)

(188)

Gold nanoparticles@DNA-Gd(III) 13 nm 16.9 (1.41 T)

DNA-Gd(III) 8.7 (1.41 T)

Gd(III)-loaded dendrimer 26.0 (2.0 T)

(197)

Core-encapsulated Gd(III) Lipossomes 3.8 (1.5 T)

(192)

Surface-conjugated Gd(III) Lipossomes 7.9 (1.5 T)

Gd(III) DOTA-DSPE-based Liposomal 11.8 (1.41 T)

(198)

Gd2O3-D-glucuronic acid 9.9 (1.5 T)

(199)

Source: By the author.

112

4.2 EXPERIMENTAL SECTION

4.2.1 Materials

Tetraethoxysilane (TEOS) and 3-aminopropyl)triethoxysilane (APTES) were

purchased from Gelest, S-2(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-

1,4,7,10-tetraaceticacid) (Gd-DOTA-SCN) was purchased from Macrocyclics,

tetrakis(hydoxymethyl) phosphonium chloride (THPC), Gd(NO3)3.6H2O, and chloroauric acid

(HAuCl4.3H2O) were purchased from Sigma Aldrich; anhydrous potassium carbonate

(K2CO3) from Fisher, mPEG-Thiol (MW = 10000) and Thiol-PEG-NH2 (MW = 10000) from

Laysan Bio, Inc.; NaOH from Fisher and employed without further purification. 50 nm Gold

Colloid citrate NanoXact was purchased from NanoComposix. Dulbecco’s Modified Essential

Media (DMEM) was purchased from ATCC, fetal bovine serum (FBS), Phosphate Buffer

Saline (PBS), Antibiotic-Antimycotic Solution from CellGro and RAW 264.7 macrophage

cells were purchased from Sigma-Aldrich. 200 proof ethanol was purchased from KOPTEC.

SYTOX® Red dead cell stain was purchased from Invitrogen. Aqua regia was used to clean

all glassware and stirring bars, followed by thorough rinsing with distilled water, ethanol and

Milli-Q water in the last step. Milli-Q water (18.2 MΩ.cm at 25 °C, Millipore) was used to

prepare all solutions.

4.2.3 Gd-embedded Au nanomatryoshkas (Gd-NM) synthesis

The synthesis of Gd-NM developed in this study was adapted from the previously

reported synthesis of fluorescent dye encapsulated NM by Ayala-Orozco et al.(200) The 50

nm Au nanoparticles were initially coated with an amorphous SiO2 layer by the condensation

of tetraethyl orthosilicate in an alkaline medium. Briefly, 21 mL of 50 nm Au colloid solution

(0.05 mg/mL) was mixed with 180 mL of 200 proof fresh ethanol under magnetic stirring and

subsequently, 1.8 mL of ammonium hydroxide were quickly added followed by 36 µL 10%

TEOS solution in ethanol and 21 µL of 10% APTES solution in ethanol. The reaction vessel

was sealed and allowed to proceed for 75 min at room temperature under vigorous stirring and

then stored in the refrigerator under gentle stirring at 4 °C. After 165 min (total reaction time),

a mixture previously prepared containing a Gd(III) chelate was added. The mixture contains:

15 µL of Milli-Q H2O, 5.5 mg of Gd(III)-DOTA-SCN, 125 µL of ethanol, and 8 µL of a 20%

113

APTES ethanolic solution. This mixture was kept for at least 1 h under gentle stirring at room

temperature and protected from light. After the addition of Gd(III) chelate mixture to the

nanoparticle suspension, the system was maintained under gentle stirring at 4 ºC for a total

reaction time of 27 h. The Gd(III) ions were incorporated into the APTES-doped silica layer

of the NM by using a covalent strategy. More specifically, the SCN groups of the chelate

Gadolinium(III) S-2 (4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-

tetraaceticacid), was bound to the amine groups of the APTES.

In the following day, the Au/SiO2 nanoparticles were transferred into a pre-washed

(200 proof ethanol) dialysis membrane (Spectra/Por 6, MWCO = 10000, Spectrum Labs) and

dialyzed in 1 gallon of 200 proof ethanol for 16 h at room temperature to remove the

remaining free reagents. The cleaned nanoparticle suspension was cooled to 4ºC for at least 2

h and then centrifuged 35 min at 1700 g. The supernatant was removed and the pellets were

dispersed in 3 mL of 11 mg/mL Gd(NO3)3 aqueous fresh solution. The system was then

sonicated for 10 min, kept at room temperature for 100 min, and sonicated again for 10 min.

The suspension was centrifuged at 1800 g for 25 min and the pellet was dispersed in 2 mL of

water, and then centrifuged one more time at 1500 g for 25 min. The cleaned pellet was

dispersed in 1 mL of water and sonicated for 5 min. Gd(NO3)3 was added to reload the Gd(III)

ions onto the chelate, since the extremely basic conditions, such as high ammonia

concentrations, and multiple rinsing steps can remove some ions from the chelates.

The Au/SiO2/Au nanoparticles were prepared by decorating the silica coated

nanoparticles with small gold colloid (2-3 nm) fabricated by the method reported by Duff et

al.(73) Briefly, 1 mL of the Gd(III) doped silica coated gold nanoparticle suspension was

mixed with 600 µL of 1 M NaCl and 40 mL of Duff gold colloid (1-2 nm). The precursor

particles were left unperturbed for 24 h at room temperature, followed by sonication and

centrifugation (950 g, 30 min) to remove the excess of Duff gold colloid. The pellet was

dispersed in 2 mL of Milli-Q water and centrifuged two more times at 800 g for 20 min. The

final precipitate was dispersed in 500 µL of Milli-Q H2O and was called the seeded precursor.

The formation of a continuous metallic shell around the seeded precursor was performed by

mixing 3 mL of plating solution (1% HAuCl4- K2CO3 solution previously prepared) with 20-

40 µL of the seeded precursor and 15 µL of formaldehyde, under a fast shaking for 1 min.

The color of the solution changed from reddish to purple upon the formation of the gold outer

shell.

114

4.2.4 Polyethylene glycol (PEG)-functionalization of Gd-NM (Gd-NM-PEG)

To provide chemical stability, the Gd-NM were functionalized with a thiolated mPEG

layer as follows: 3 mL of ~3x1010

particles/mL aqueous nanoparticles were mixed with a

freshly prepared mPEG-Thiol (MW = 10,000, Laysan Bio, Inc) aqueous solution under

sonication to a final solution concentration of 100 µmol/L. The system was sonicated for 30

min and then kept under gentle stirring and protected from light for 12 h. The system was then

centrifuged at 700 g for 20 min and re-suspended in 500 µL of Milli-Q water. For the in vitro

studies, after conjugation with mPEG-Thiol solution, the mPEG coated Gd-NM solution was

diluted in 1 mmol L-1

phosphate buffer pH = 7.4, sterilized by filtration

through a 0.22 µm filter (Whatman) and then concentrated via centrifugation (700 g, 20 min)

to the final concentration of ~1011

particles/mL.

4.2.5 GdPEG-coated gold nanomatryoskas (GdPEG-coated NM) synthesis

Bare gold nanomatryoshkas were also synthesized and then functionalized externally

with Gd(III) chelate for comparasion. Initially, bare NM were synthesized. Briefly, 21 mL of

0.05 mg/mL 50 nm Au colloid were mixed with 180 mL of 200 proof ethanol, 1.8 mL of 28-

30% ammonium hydroxide, 36 µL 10% TEOS solution in ethanol, and 36 µL of 10% APTES

in ethanol. The reaction was allowed to proceed for 65 min at room temperature under

vigorous stirring and then stored in the refrigerator at 4 °C for 22 h. The following day, the

Au/SiO2 nanoparticle suspension was dialyzed in 200 proof ethanol for 16 h at room

temperature. The nanoparticle suspension was cooled to 4ºC, centrifuged at 1500 rcf for 35

min and re-dispersed by sonication in a total volume of about 3 mL of ethanol. The precursor

Au silica nanoparticles were coated with small gold colloid (2-3 nm) by mixing 3 mL of

Au/SiO2 nanoparticles with 40 mL of Duff Au colloids, and 600 µL of 1 M NaCl solution.

The mixture was sonicated for 20 min and incubated for 24 h at room temperature following

which they were sonicated for 15 min and centrifuged at 900 g for 25 min. The pellet was

dispersed in 2 mL of Milli-Q water and centrifuged two more times at 800 g for 20 min, the

supernatant removed and the final pellet was dispersed in 500 µL H2O. A continuous gold

shell was grown around the Au/SiO2 nanoparticles following the same procedure of Gd-NM.

The as-prepared NMs were centrifuged at 700 g for 20 min and dispersed in 3 mL of

H2O. The system was sonicated for 10 min and then a solution containing Thiol-PEG-NH2

was added to a final concentration of 100 µmol L-1

. After 30 min of sonication and overnight

115

incubation at 4ºC, the system was centrifuged at 700 g for 20 min and resuspended in 3 mL of

Milli-Q water. In the next step, the NH2-PEG-NM was incubated with 300 µL, of 0.01 mg/mL

Gd-DOTA-SCN aqueous solution and 20 µL 1 N NaOH solution. The mixture was sonicated

for 30 min and incubated overnight at room temperature under gentle stirring. The Gd(III)

chelates bind covalently to the NH2 group of the PEG molecules. The excess of Gd(III)

chelate and PEG molecules were removed by centrifugation twice at 700 g for 20 min

followed by dispersal with Milli-Q to a final concentration of ~1011

particles/mL.

4.2.6 Calculation of Nanomatryoshkas Concentration

The concentration of the nanomatryoshkas was determined by the following equation

derived from beer's law:(10)

(4.5)

where A = experimental extinction at the peak plasmon resonance

= theoretical extinction cross section taken from Mie theory

l = path length of the cuvette (1 cm)

The calculation of the extinction cross section by Mie theory was discussed in Chapter

2. The constant parameters used in the calculation were: dielectric constant of the SiO2 of 3.0,

dielectric constant of the medium (H2O, 1.77), dielectric constant of gold, and theoretical

extinction efficiency of 𝑄 = 5.53. The calculated value of the extinction cross section for the

typical Gd-NM used in this study with dimensions of [r1, r2, r3] = [25, 38, 53] nm was =

6.89x10-10

cm2. This value was substituted in Equation 4.5 and the concentration of particles

was calculated based on the experimental extinction (A) measured in a Cary 5000 UV-VIS-

NIR Varian spectrophotometer.

4.2.7 Characterization

The size and morphology of both Gd-NM and GdPEG-coated NM in each step of the

synthesus were evaluated by transmission electron microscopy (TEM) using a JEOL 1230

operating at 80 kV. For this, one drop of the diluted samples were deposited on a carbon film

116

copper grid 200 mesh (Ted Pella, Inc.) and dried at room temperature. For a further detailed

investigation, the particles were also imaged in a high resolution JEOL 2010 TEM operating

at 200 kV.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the

interactions between the Gd-DOTA-SCN and the amine groups from the APTES-doped silica

layer. For this, FTIR spectra of Gd-DOTA-SCN solution, silica-coated Au colloids and

Gd(III)-embedded silica-coated Au colloids deposited on silicon substrates were obtained

with a resolution of 4 cm-1

in absorbance mode using a Vertex 80v FTIR Spectrometer

(Bruker).

The investigation of the optical properties was performed by ultravileta-visivel-near

infrared (UV-VIS-NIR) spectroscopy using a Cary 5000, Varian spectrometer. Dynamic light

scattering (DLS) was used to measure the in situ hydrodynamic size distribution of the

nanostructures in suspension in each step of the synthesis process. The analyses were always

performed in triplicate at room temperature using a Malvern Nano-ZS spectrometer (Malvern

Instruments, UK). The surface change around the particles due the functionalization was

evaluated by zeta potential. The measurements were performed in triplicate at room

temperature using a Malvern Nano-ZS spectrometer (Malvern Instruments, UK).

4.2.8 Inductively Coupled Plasma Mass Spectroscopy (ICP-MS)

The concentration of Gd(III) in the particles were determined by ICP-MS, which is

able to detect metals in a sample at concentrations as low as 1 ppm. The technique consists in

two primary components: the inductively coupled plasma (ICP) and the mass analyser (MS).

The samples are digested in acid solution and introduced into ICP using a nebulizer. The ICP

is an electrically conductive Argon gas at very high temperatures (~10000 K) which

evaporates the solvent and atomizes and ionizes the sample before it is analyzed in the MS.

The MS used was a quadrupole mass spectrometer that consists of two parts of electrically

conductive rods connected in series. The charged samples travel throught the quadrupole at a

rate that is determined by the mass to charge (m/z) ratio of the ionized atoms. Only the

selected m/z ratio will reach ion detector with all other ions colliding with the quadrupole rods

allowing sensitive and selective detection. The measurements were performed in a Perkin

Elmer Nexion 300. First, the Gd-NM or GdPEG-coated NM samples were mixed with

concentrated aqua regia (HNO3:HCl 1:3) solution overnight. The resulting solution was

117

diluted by 500 times with a solution consisting of 1% v/v aqua regia, and 2% v/v HNO3

solution was used for ICP-MS analysis. The Gd(III) concentration was determined using a

calibration curve made with Gadolinium ICP/DCP standard solution (Fluka).

4.2.9 Gd(III) release

The release of Gd(III) from both Gd-NM-PEG and GdPEG-coated NM was

investigated. For this, the concentration of Gd(III) in each system was determined by ICP-

MS. Then, the particles were dispersed in PBS buffer and serum media (10% FBS) and kept

at 37ºC for 48 h. After that, the particles were centrifuged at 1000 g for 15 min, and the

concentrations of Gd(III) in both the supernatant and the pellet were determined by ICP-MS.

4.2.10 MRI Analysis

The MRI relaxation measurements were performed on a 4.7 T Biospec spectrometer

(Bruker Biospin MRI, Billerica, MA) with a 30 cm bore horizontal imaging system, using

imaging gradients with an inner diameter of 60 mm and a volume resonator with 35 mm inner

diameter. Dilutions of the particle solution were sealed in 200 µL PCR tubes and placed in a

holder. Spin-lattice (T1) relaxation times were measured using a RARE (Rapid Acquisition

Relaxation Enhanced) variable TR sequence (TE = 10.5 ms; with 10 TRs from 15000 to 400

ms). Spin-spin (T2) relaxation times were measured using a multi-echo sequence (TEmin = 10

ms, with 10 ms echo spacing over 30 echoes; TR = 5000 ms). All images were acquired with

matching slice geometry (1 mm axial sections, 32 mm x 32 mm field-of-view over a 256 x

256 image matrix). Relaxation time constants for each sample were measured by fitting signal

decay curves to a standard model in ParaVision 5, the operating software for the Biospec

platform.

4.2.11 Cytotoxicity

The RAW 264.7 macrophage cells (Sigma-Aldrich) were cultured in Dulbecco’s

Modified Essential Media (DMEM, ATCC) supplemented with 10% fetal bovine serum

(FBS, CellGro) and 1% Antibiotic-Antimycotic Solution (CellGro) and maintained at 37°C

with 5% CO2. This type of cell has been included in nanomaterial toxicity investigations as an

118

inflammatory cell type.(201) Cells were initially seeded in 6-well plates at a density of 5x105

cells/well and incubated for 24 h prior the introduction of Gd-NM-PEG and GdPEG-coated-

NM. The particles were added to the serum containing media in a NM:Cell ratio of 3000:1

and 10000:1. After 20 h of incubation with NMs, the media was removed. Cells were rinsed

with PBS solution and detached from the plate using a sterile cell scrapper and washed by

centrifugation twice in buffer to remove the excess of media. The cells from each individual

well were dispersed in a final volume of 500 µL of PBS, stained with SYTOX® Red dead cell

stain, and analyzed by flow cytometry using an Ex: 635 nm laser. The measurements were

performed on an LSRFortessa X20 cell analyzer (BD Biosciences).

4.2.12 Fluorescence Imaging

Differential interference contrast (DIC) and confocal images were taken to investigate

the Gd-NM-PEG cellular internalization. Briefly, the cells were cultured for 24 h in a µ-Slide

8 well (Ibidi) and, then incubated with the particles. After 20 h, the cells were rinsed three

times with PBS and fixed with a 4% paraformaldehyde solution. The cells were washed twice

with PBS and incubated with a Hoechst 33342 solution to stain the cell nucleus. After an

incubation of 10 min protected from light, the cells were rinsed twice with PBS. All images

were collected on a Nikon A1-Rsi Confocal microscope and the particles were imaged in the

reflection mode (Excitation and Emission at 640 nm).

4.2.13 Preparations of Cells for MRI

RAW 267.4 macrophages cells ~5x105 cells/well were seeded in a 6 well plate for 24

h, then the media was removed and the cells were incubated with media containing Gd-NM-

PEG for 20 hours. The cells were rinsed in PBS three times to remove the excess of non-

internalized nanoparticles, detached from plate using a cell scrapper and centrifuged. The

cells were dispersed in a final volume of 200 µL PBS with a final concentration of ~5.2x107

cells/mL. The T1-weighted MR images of the cells were obtained at 4.7 T and 25ºC.

119

4.2.14 Phantom preparation for MRI

Phantoms of 1% agarose gel were prepared in 200 μL centrifuge tubes. Briefly, the

agarose gel was heated until the complete solubilization, and the Gd-NM-PEG was

subsequently transferred to the warm agarose and allowed to cool in ambient conditions to

form the gel, ensuring that no air bubbles were formed during cooling. The final Gd(III)

concentrations were: 0.6, 2.5, 5, 10, and 20 μM, correspondent to a 0.14, 0.6, 1.14, 2.3, and

4.6x109 in number of Gd-NM-PEG The final volume was 100 μL for all concentrations. The

T1-weighted MR images of the phantoms were obtained at 4.7 T and 25ºC.

4.3 RESULTS AND DISCUSSION

Gd-NM nanoparticles were fabricated as shown in Figure 4.2A. Transmission

electron microscope images corresponding to each stage of the synthesis are shown in Figure

4.2Bi-iv. Au nanoparticles of radius 50 ± 4 nm (Figure 4.2Bi) were initially coated with a

~21 nm amorphous silica layer doped with Gd(III) chelates (Figure 4.2Bii). The

isothiocyanate (N=C=S) group of the Gd-DOTA-SCN chelate is believed to bind covalently

to the NH2 group of the APTES molecules within the silica network. This results in the

formation of a thiourea (NH-(C=S)-NH) bond that was monitored by FTIR measurements

(Figure 4.3). As a result of the formation of this bond, the peak at 2100 cm-1

(attributed to the

N=C=S vibration in the Gd(III) chelate molecules) disappears and the peak at 1100 cm-1

(attributed to the C=S bound in the thiourea) appears after incorporation of the chelate into the

silica layer.(202)

In the next step, Gd-DOTA-SCN-doped APTES silica-coated Au colloid were

incubated with Gd(NO3)3 to reload the Gd(III) ions, since the extremely conditions, such as

high ammonia concentrations during the silica growth, can take out some ions from the

chelates. The Gd(III) loaded within the SiO2 layer before and after incubation with Gd(NO3)3

was examined by high resolution TEM (Figure 4.4). The TEM images reveal that the

Gd(NO3)3 etches the silica layer and promotes some darkening of the contrast as well, which

is probably due the incorporation of many Gd(III) ions. The Gd(NO3)3 solution may also

affect the porosity and accumulation of H2O molecules in the silica layer.

120

Figure 4.2 - (A) Schematic representation of the MRI active nanomatryoshka synthesis showing the stepwise

synthesis process: the 50 nm diameter gold colloids are coated with Gd(III) chelates embedded in a

SiO2 shell and then a continuous Au shell. (B) TEM images corresponding to each step in the

process; scale bars are 100 nm.

Source: By the author.

After growth of the Gd(III)-embedded silica layer, 2-3 nm diameter Au colloidal

nanoparticles were attached to the outer surface of the silica layer. At this stage we refer to the

nanoparticles as the “seeded precursor”. The ultrasmall Au nanoparticles serve as nucleation

sites for the electroless plating of the outer Au shell layer (Figure 4.2Biii). This results in

Gd-NMs with average dimensions of [r1, r2, r3] = [25, 38, 53] nm (Figure 4.2Biv). From the

TEM image, we can see that this multistep synthesis produces stable, monodisperse Gd-NM

with a continuous outer Au layer. The final Au shell also allows direct conjugation of

polymers and biomolecules to the nanoparticle surface.

Gd(III) chelate 2 nm Au NP

A

B

TEOS, APTES

CH2O

Au colloid Gd-doped SiO2/ Au colloid Seeded precursor Gd-NM

Gd(NO3)3 Au3+

i ii iii iv

100 nm 100 nm 100 nm 100 nm

121

Figure 4.3 - (A) FTIR spectra of (i) Au@SiO2-APTES, (ii) Gd-DOTA-SCN, and (iii) Gd-doped SiO

2-coated Au

colloid. The disappearance of the 2100 cm-1

peak, attributed to the N=C=S vibration, is an

indicative of the bond between Gd-DOTA-SCN and the amine groups from APTES (B)the

chemical reaction showing the bond formation. The table shows the FTIR peak assessments

corresponding to each spectrum.

Source: By the author.

Figure 4.4 - High resolution TEM images of: (A) SiO2-coated Au colloid and (B) Gd-doped SiO2 coated Au

colloid.

Source: By the author.

To optimize the MRI contrast of the Gd-NMs, the concentration of Gd(III) per particle

was varied by modifying the Gd chelate concentration and reaction time. The MRI relaxivity

as a function of Gd(III) concentration is shown in Figure 4.5A. The relaxivities r1 were

Au@SiO2AP

TES

Gd-DOTA-

SCN

Au@SiO2-APTS-

Gd-DOTA-SCN

Peak

assignments

1580 --- 1607 NH2 (def)

1390 --- 1396 C-N (str)

1089 --- 1084 Si-O-Si (str)

--- 2100 --- N=C=S (str)

--- 1626 1607 COO- (str)

--- 1396 1396 COO- (str)

--- --- 1100 C=S (str)4000 3000 2000 1000

iii

i

Ab

so

rba

nce

(a

.u.)

Wavenumber (cm-1)

ii

A

B

10 nm10 nm

A B

122

calculated from the 1/T1 versus Gd(III) concentration data. The T1 relaxivity curves for three

representative concentrations are shown in Figure 4.6. We observed that at low Gd(III)

concentrations, the MRI relaxivity r1 increased with increasing Gd(III) concentration per NM.

For example, the relaxivity of 0.7x105 Gd(III) ions/NM was 8 mM

-1 s

-1 and increased until a

maximum of 14.6 mM-1

s-1

at 2.5x105 Gd(III) ions/NM was reached. As the amount of Gd(III)

per NM was increased further, to 8.2x105 Gd(III)/NM, the relaxivity decreased to 5.3 mM

-1

s-1

.

This type of quenching effect has been observed in other nanoparticle-based Gd(III)

systems developed for MRI.(175, 203, 204). In these cases, quenching was attributed to the

packing of Gd(III) into a limited volume, which could restrict the access of H2O molecules to

the coordination sphere of the Gd(III). However, as we will discuss shortly, our results

indicate that, in the case of Gd-NMs, the inner coordination sphere H2O molecules do not

likely play a significant role in the enhanced T1 relaxivity. It has also been proposed that

systems with an excessive payload of Gd(III) may lead to a disproportionate weight of

T2 effects, which might have some negative effects on T1 weighted images(175, 204-206). We

did observe an increase in T2 relaxivity by increasing the number of Gd(III) per nanoparticle.

For example, for a system with 2.3x105 Gd(III) per nanoparticle, the r2 was nominally 54.7

mM-1

s-1

, while for 4.7x105 it was 93.6 mM

-1 s

-1. This increase in T2 relaxivity has, in other

nanoparticulate systems, been attributed to the geometric confinement of Gd(III) with

increased dipolar interaction between neighboring Gd(III)-Gd(III) ions and/or Curie spin

relaxation.(207)

Figure 4.5 - (A) The r1 relaxivity of Gd-NM as a function of the number of Gd(III) per NM measured at 4.7 T.

(B)The r1 relaxivities of Gd(III)-DOTA (for comparison), Gd(III)-NM, and Gd(III)-NM-PEG;

error bars represent standard deviation of three independent experiments at 25ºC. (C) Thermal

variation of T1 for Gd-NM-PEG at 25ºC (blue, r1 = 14.6 mM-1

s-1

) and 37ºC (red, r1 = 16.1 mM-1

s-

1). The r1 values were extracted from the slopes.

Source: By the author.

0.000 0.005 0.010 0.015 0.020

0.3

0.4

0.5

0.6

0.7 25C

37C

1/T

1 (

s-1

)

Gd(III) concentration (mM)

0 2 4 6 8

6

8

10

12

14

x105

Re

laxiv

ity (

mM

-1 s

-1)

Number of Gd(III) per NM

A B C

0.000 0.005 0.010 0.015 0.020

0.4

0.5

0.6

0.7

0.8

r1

= 14.0 mM-1s-1

Gd-DOTA-SCN chelate

Gd-NM

Gd-NM-PEG (5k)

Gd-NM-PEG (10k)

1/T

1 (

s-1)

Gd(III) concentration (mM)

r1

= 6.0 mM-1s-1

r1

= 16.1 mM-1s-1

r1

= 18.0 mM-1s-1

r1 = 14.6 mM-1s-1

r1 = 16.1 mM-1s-1

123

Figure 4.6 - T1 (longitudinal) rate of Gd-NM for: (A) 0.8x10

5

, (B) 2.7x105

, and (C) 8.2x105

numbers of Gd(III)

ions per particle at 4.7 T and 25ºC. The r1 values were extracted from the slopes.

Source: By the author.

The nanoparticle relaxivity can also be affected by nanoparticle aggregation, which

can in turn be influenced and controlled by nanomatryoshka surface chemistry. To prevent

aggregation, the Gd-NMs were functionalized with thiolated PEG molecules of 5,000 and

10,000 MW. PEG functionalization also improves NM dispersion in media, and is known to

increase circulation time in vivo.(208) The increasing in the hydrodynamic diameter and the

reduction of zeta potential to close to zero shown in Figure 4.7 confirm the coating of the

particles. The Gd-NM relaxivity increased significantly with PEG functionalization, from

14.0 mM-1

s-1

to 16.1 and 18.0 mM-1

s-1

for 5 and 10 kDa, respectively (Figure 4.5B). We can

infer from this increased relaxivity that the presence of PEG molecules on the surface of the

nanoparticles facilitates the approach of water protons to the NM surface, closer to the

internally encapsulated Gd(III) ions. The diffusion of water in the proximity of the Gd(III) is

known to play an important role in the enhancement of proton relaxivity.(195, 209-210) For

example, Cho et al evaluated the effect of the surface coating of Gd2O3 nanoparticles in the r1

relaxivity.(211) They showed that the surface modification with poly(acrylic acid) enhanced

the r1 relaxivity, it was attributed to the modulation of water molecules diffusion by the

surface coating. Similarly, Sun et al. attributed the longitudinal relaxivity enhancing, among

others, to an increase number of outer coordinated water molecules via entrapment by the

polysaccharide for the polysaccharide-Gd-DTPA.(212)

0.000 0.005 0.010 0.0150.3

0.4

0.5

0.6

r1 = 14.5 mM-1s-1

1/T

1 (

s-1

)

Gadolinium concentration (mM)

0.000 0.005 0.010 0.0150.3

0.4

0.5

0.6

r1 = 9.7 mM-1s-1

1/T

1 (

s-1

)

Gadolinium concentration (mM)

0.000 0.005 0.010 0.0150.3

0.4

0.5

0.6

r1 = 5.3 mM-1s-1

1/T

1 (

s-1

)

Gadolinium concentration (mM)

A B C

124

Figure 4.7 - (A) Dynamic Light Scattering and (B) Zeta potential for Gd-NM before and after PEG surface

functionalization.

Source: By the author.

Besides Gd(III) concentration and surface functionalization, temperature can also

strongly affect the relaxivity, which can have a major impact in vivo.(213) We compared the

relaxivity measurements of Gd-NM-PEG at 37 °C and at room temperature (Figure 4.5C).

The relaxivity was found to increase from r1 = 14.6 mM-1

s-1

when the ambient temperature of

the nanoparticles was increased from room temperature to r1 = 16.1 mM-1

s-1

at physiological

temperature. This effect has been reported for other nanostructured gadolinium system, such

as liposomal structures.(204) However, the opposite is observed for small molecular weight

Gd(III)-based compounds, i.e., the relaxivity decreases with increasing the temperature.(214)

This is expected because the rotational correlation time is a limiting parameter parameter for

small molecules.(214)

In the following experiments, Gd-NM-PEG was compared to a “control” NM, where

Gd(III) was not incorporated inside the nanoparticle but was instead attached to the outside of

the NM through conjugation of Gd-DOTA-SCN to aminated PEG linker molecules. Figure

4.8 shows the schematic representation of the bare NM synthesis and functionalization. The

increase in the hydrodynamic size and the positive zeta potential confirm the functionalization

of NM with the Thiol-PEG-NH2 (Figure 4.9). After conjugation with Gd-DOTA-SCN, the

hydrodynamic diameter had a small decrease, which is probably related to the removal of

polymer excess during the several steps of centrifugation. The negative zeta potential

confirms the conjugation with the Gd-DOTA-SCN (Figure 4.9B). These nanoparticles will be

referred as GdPEG-coated-NMs.

Since the presence of biologically active molecules in serum can impact the stability

of complex nanostructures,(215) the amount of Gd(III) released from both types of

0

50

100

150

200

Hyd

rod

yn

am

ic d

iam

ete

r (n

m)

Gd-NM PEG-Gd-NM

-30

-20

-10

0

10

PEG-Gd-NMGd-NM

Ze

ta P

ote

ntia

l (m

V)

BA

125

nanoparticles was measured after incubation at 37 °C for 48 h in both 10% fetal bovine serum

(FBS) and in H2O by ICP (Figure 4.10). For the Gd-NM-PEG system, only 2.7% of Gd(III)

was released, while for the GdPEG-coated-NM suspended in H2O 48.6% of the Gd(III) ions

dissociated from the nanocomplex (Figure 4.10). In fetal bovine serum, only 5.1% Gd(III)

was released from Gd-NM, while 97.5% of the Gd(III) dissociated from the GdPEG-coated-

NM. These results clearly indicate that the confinement of Gd(III) between the Au core and

the Au shell prevents the release of Gd(III) from the nanoparticle under these conditions.

Figure 4.8 - (A) Schematic representation of (A) the regular nanomatryoshkas synthesis and (B) the

functionalization of the NM with Gd(III) chelate (GdPEG-coated-NM).

Source: By the author.

Figure 4.9 - (A) Dynamic Light Scattering and (B) Zeta potential for regular NM before and after conjugation

with PEG-NH2 and Gd chelates (GdPEG-coated-NM).

Source: By the author.

Gd(III) chelate

NaOH

SH-PEG-NH2

2 nm Au NP

A

TEOS, APTES

CH2O

Au colloid SiO2-coated Au colloid Seeded precursor NM

Au3+

NM

B

NM-PEG-NH2 Gd-coated NM

-40

-20

0

20

40

Zeta

pote

ntial (m

V)

0

50

100

150

200

Hyd

rodyn

am

ic d

iam

ete

r (n

m)

NM PEG-NM GdPEG-NM NM PEG-NM GdPEG-NM

BA

126

Figure 4.10 - Percentage of Gd(III) ions released from Gd-NM-PEG, where the Gd(III) is located inside the NM

which is then functionalized using PEG, versus GdPEG-coated NM, where the Gd(III)-DOTA-

SCN is attached outside the NM through a thiol-PEG-amine linker, after 48 h of incubation at

37ºC in 10% FBS (red) and H2O (blue). Gd(III) concentration was quantified using ICP-MS.

Source: By the author.

The physiological environment also play an important role in the MRI efficiency of

the contrast agent.(213) In this way, the impact the serum dispersion (10% FBS) on the

relaxivity and the plasmon resonance of both Gd-NM-PEG and GdPEG-coated-NM (Figure

4.11) were compared. Following 24 hr incubation, little change in either r1 relaxivity or

extinction spectrum for Gd-NM-PEG was observed, while the GdPEG-coated-NM showed

changes in both relaxivity and in extinction spectrum (Figure 4.11). Since particle

aggregation (176) was not observed (Table 4.3), the release of the Gd(III) ions (Figure 4.10)

may be the responsible for inducing the observed changes in the GdPEG-coated NM case.

Also, the increasing in the hydrodynamic diameter for GdPEG-coated NM indicate

nonspecific protein binding, which is probably due the high surface charge originated from

the Gd(III) chelates on the particle surface.

Gd-NM-PEG GdPEG-coated-NM0

20

40

60

80

100

120

% o

f G

d(I

II)

ion

s r

ele

ase

d

in FBS

in H2O

127

Figure 4.11 - (A) T1 (longituginal) rate and (B) extinction spectra for Gd-NM-PEG in water (blue) and 10%

serum (red). (C) T1 (longituginal) rate and (D) extinction spectra GdPEG-coated NM in water

(blue) and 10% serum (red). The T1 measurements were performed at 4.7 T and 25ºC.

Source: By the author.

Table 4.3 - Hydrodynamic diameter of Gd-NM-PEG and GdPEG-coated NM in water and after dispersion in

culture media (FBS 10%) measured by dynamic light scattering (DLS).

Sample In water In FBS 10%

Gd-NM-PEG 167.0 ± 2.7 nm 168.1 ± 2.3 nm

GdPEG-coated NM 152.7 ± 2.1 nm 161.4 ± 2.4 nm

Source: By the author.

To better understand how this specific geometry impacts the Gd-NM relaxivity, we

examine how the relaxivity is modified at two stages of Gd-NM synthesis. The comparative

relaxivity study was performed for the seeded precursor (Figure 4.12A), the complete Gd-

NM-PEG (Figure 4.12B), and the GdPEG-coated-NM (Figure 4.12C). For each structure,

this information is accompanied by the corresponding extinction spectrum (Figure 4.12D, E,

F) and TEM image (Figure 4.12G, H, I). All measurements were performed at 4.7 T and

0.000 0.005 0.010 0.0150.3

0.4

0.5

0.6

1/T

1 (

s-1)

Gadolinium concentration (mM)

0.000 0.005 0.010 0.0150.3

0.4

0.5

0.6

1/T

1 (

s-1)

Gadolinium concentration (mM)

400 600 800 1000

Extin

ction

(a.u

.)

Wavelength (nm)

A B

C D

400 600 800 1000

Extin

ction

(a.u

.)

Wavelength (nm)

128

25ºC. The r1 values were calculated to be 21.5 mM-1

s-1

, 17.9 mM-1

s-1

, and 13.6 mM-1

s-1

for

the seeded precursor, Gd-NM-PEG, and GdPEG-coated-NM, respectively. We find that the

addition of the Au outer shell to the seeded precursor nanoparticle decreases its relaxivity,

since the growth of a continuous metal shell layer limits the access of water molecules to the

coordination spheres of the Gd(III) within the interior silica layer. However, the relaxivity of

the GdPEG-coated-NM particles was lower than the one observed for the Gd-NM-PEG. Two

effects contribute to this reduced relaxivity: (1) the Gd(III) concentration per nanoparticle for

the Gd-coated-NM nanoparticles was slightly lower than for the Gd-NM-PEG, 3.9 ± 0.7x104

vs. 1.5 ± 0.4x105 Gd(III)/nanoparticles, and (2) additional confinement effects due to the

incorporation of the Gd(III) into the silica layer in the Gd-NM-PEG case. Taking into the

number of Gd(III) per nanoparticle, the relaxivity per nanoparticle was calculated to be

approximately 2.7x106 mM

-1 s

-1 and 5.3x10

5 mM

-1 s

-1for Gd-NM-PEG and GdPEG-coated-

NM, respectively. For comparison, molecular chelate Gd(III) contrast agents in current

clinical use typically have relaxivities r1 of nominally 4 mM-1

s-1

per Gd(III) at 1.5 T and 37

°C.(216) As shown in the T1-weighted MRI images (Insets of Figure 4.12A, B, and C), the

Gd-NM-PEG produced consistently brighter images than the GdPEG-coated NM, supporting

its use as a T1 agent for MRI applications.

129

Figure 4.12 - T1 (longitudinal) rate versus Gd(III) concentration at 4.7 T for (A) seeded precursor, (B) Gd-NM-

PEG, and (C) GdPEG-coated-NM; and the corresponding (D,E,F) extinction spectra and (G,H,I)

TEM images; (scale bar are 50 nm). (Insets A-C: T1 weighted MR images).

Source: By the author.

The enhanced relaxivities of Gd(III)-containing nanostructures relative to molecular

chelates can be attributed not only to the additive effect of many Gd(III) centers, but also to

their slower rotational motion.(23,217) According to the Solomon-Bloembergen-Morgan

(SBM) theory of paramagnetic relaxation, the main factors that affect the relaxivity of

Gd(III)-based contrast agents are molecular diffusional and rotational times, number of

coordinated water molecules, water proton residency lifetime and water exchange rate.(218)

In general, a decrease in the molecular diffusion and rotation times leads to an increased T1

400 600 800 1000

Extin

ctio

n (

a.u

.)

Wavelength (nm)

0.00 0.01 0.020.2

0.4

0.6

0.8

1.0

r1 = 13.6 s-1 mM-1

1/T

1 (

s-1

)

Gd(III) concentration (mM)

0.00 0.01 0.020.2

0.4

0.6

0.8

1.0

r1 = 21.5 s-1 mM-1

1/T

1 (

s-1

)

Gd(III) concentration (mM)

0.00 0.01 0.020.2

0.4

0.6

0.8

1.0

r1

= 17.9 mM-1s-1

1/T

1 (

s-1

)

Gd(III) concentration (mM)

400 600 800 1000

Extin

ction

(a.u

.)

Wavelength (nm)

400 600 800 1000

Extin

ction

(a.u

.)

Wavelength (nm)

B

C

A D

E

F

G

H

I

130

relaxivity, especially at low magnetic fields.(23,213) Therefore, the incorporation of Gd(III)

into nanostructures decreases its molecular tumbling rate, and, consequently, reduces its

diffusional and rotational correlation time, increasing relaxivity.(218) Besides the enhanced

T1 relaxivity, another important advantage of nanostructured systems is the increased

accumulation of carriers in target tissue, for example, exploiting the enhanced permeability

and retention effect in tumors,(219) which increases the local concentration of the contrast

agent in the nanoparticle case.(218)

It is worth emphasizing that our measurements were performed at 4.7 T. Higher

magnetic fields provide not only a greater signal to noise, but also a higher spatial resolution

and reduced acquisition times.(213) MRI scanners with higher fields, such as 3, 4.7 or even

9.4 T, have been in use clinically. However, the T1 relaxivity of molecular Gd(III) compounds

typically decreases as the magnetic field increases.(213, 220) The effect of the magnetic field

on the relaxivity was shown to be more pronounced for slowly tumbling molecules than for

rapidly tumbling molecules.(213) Small molecules such as Gd-DTPA show a modest

decrease in r1 with field; however, for gadolinium chelates bound to serum albumin, the

relaxivity decreases from 24.3 mM-1

s-1

at 1.4 T to 11.2 mM-1

s-1

at 4.7 T. Similarly, Gd(III)-

chelate-functionalized gold nanostars showed a r1 relaxivity of 54.7 mM-1

s-1

at 1.41 T that

was reduced to 9.4 mM-1

s-1

at 7 T.(32) Therefore, it is a major challenge to develop MRI

contrast agents that have good r1 and can also be used at higher magnetic fields. Gd-NMs

appear to be an attractive candidate for higher-field MRI applications.

The MRI contrast mechanism depends not only on the T1 or T2 relaxation rate, but

also on the proton density in the surrounding medium and the distance between the proton

nuclei and the Gd(III). To investigate the effect of the distance between the proton nucleus

and the encapsulated Gd(III) on the T1 MRI mechanism, Gd-NM-PEG structures with two

different outer Au layer thicknesses were fabricated and investigated. The T1 longitudinal

rates at 4.7 T for the Gd-NM-PEG nanoparticles with the thinner outer shell ([r1, r2, r3] = [25,

38, 56 nm]), and the thicker shell ([r’1, r’2, r’3] = [25, 38, 73 nm]) are shown in Figure 4.13.

For the thinner shell (blue), the r1 relaxation was 12.9 mM-1

s-1

, while for the thicker shell

(red), a significant decrease in r1 relaxation, to 1.2 mM-1

s-1

(Figure 4.13A). A dramatic

reduction in T1 contrast for the Gd-NM-PEG with the thicker outer layer was observed

(Figure 4.13B). In the case of the thinner Au shell, small pinhole defects in the outer layer

may be present that would slightly increase the H2O proton interaction with the internal

Gd(III), enhancing the r1 relaxitivity in this case. However, we would anticipate that this

enhancement could be no more than the 20% increase observed in relaxivities when

131

comparing the Gd-NM-PEGs with the seeded precursor nanoparticles, which represent an

extremely porous outer Au layer (Figure 4.12). The corresponding TEM images and

extinction spectra of the thin-layer and thicker-layer Gd-NM-PEG are shown (Figure 4.13B-

C). The shifts in the extinction spectra from 747 nm to 705 nm and from 550 nm to 560 nm

correspond to the reduced interlayer plasmon coupling resulting from increasing the

thickness of the outer Au shell layer.(221-222)

To further investigate the observed MRI enhancement of the Gd-NM geometry,

Solomon, Bloembergen and Morgan (SBM) theory (28,223) was adopted to estimate the

relaxivity r1 of the Gd-NM (Equation 4.6). This analysis was performed by Hui Zhang and

prof. Dr. Peter Nordlander (Rice University).

(4.6)

where: is the intrinsic relaxivity and and are inner-sphere and outer-sphere

contributions to the total relaxivity, respectively. The intrinsic relaxivity and inner-sphere

contributions are assumed to be negligible, since the Gd(III) chelates are embedded within the

SiO2 shell where few H2O molecules are present relative to the NM surroundings. According

to SBM theory,(28) the outer-sphere contribution is given by:

(4.7)

Here C is a constant (for Gd(III) chelates it is ) and d is the

distance of closest approach of H2O molecules, here taken to be the outer Au shell thickness.

D is the sum of diffusion coefficients of bulk water and of the complex is

,(224) is the spectral density function(28), and , are, the proton and

electronic Larmor angular frequencies, respectively. For our calculations, the parameters of

Gd-DOTA were selected(28) in accordance with the experimental conditions and the

magnetic field of 4.7 T. In Figure 4.13D we use this analysis to plot the longitudinal

relaxivity r1 as a function of Au shell thickness for the Gd-NM nanoparticle geometry. The

theoretical longitudinal relaxivity decreases with increasing Au shell thickness. The

relaxivity for a Gd-NM with a 18 nm thick Au shell was measured to be while

the SBM theoretical value is just slightly smaller, ; for the 38 nm shell the

relaxivity was measured to be 1.5 mM-1

s-1

, while the theoretical value for this larger shell

thickness is 0.7 mM-1

s-1

. The results are in good qualitative agreement with the theoretical

model. The slightly larger value of the experimentally observed longitudinal relaxivity

132

relative to the theoretical analysis may indicate an enhancement due to a combination of

effects not included in the SBM model. Gd(III) is weakly ferromagnetic(225) and so are Au

nanoparticles under certain conditions.(226) Therefore, the interplay between Gd(III) and Au

spins can give rise to a small magnetization within the outer Au shell. Nearby H2O molecules

may experience additional magnetization at a reduced distance, leading to an enhanced r1

relaxivity.

Figure 4.13 - (A) Longitudinal rate at 4.7 T versus Gd(III) concentration for two Au shell thicknesses of Gd-

NM-PEG: 18 nm (blue; r1 = 12.9 mM-1

s-1

) and 38 nm (red; r1 = 1.2 mM-1

s-1

) the Gd(III)

concentration per NM was 2.3x105 in both cases, (B) T1 weighted MR images corresponding to

the concentrations shown in (A), and corresponding TEM images. (C) extinction spectra of both

Gd-NM-PEG nanoparticles as denoted in (A), and (D) the calculated longitudinal relaxivity r1

versus Au shell thickness (green) using SBM theory (a concentration of 2.3x105Gd(III) chelates

per NM is used). The blue and red dots are the corresponding experimental relaxitivities.

Source: By the author.

The cytotoxicity of Gd-NM-PEG and GdPEG-coated-NM in RAW 264.7 murine

macrophage cells was evaluated by flow cytometry (Figure 4.14). The percentage of cell

viability was calculated by normalizing the control (zero NM concentration) as 100%.

Significant differences between control cells and cells incubated with the nanoparticles were

only observed for the highest concentration of GdPEG-coated-NM (ANOVA 0.05

0.00 0.01 0.02 0.03

0.4

0.6

0.8

1/T

1 (

s-1

)

Gd(III) concentration (mM)

400 600 800 1000

Extin

ction

(a.u

.)

Wavelength (nm)

A

100 nm 100 nm

B

C D

15 20 25 30 35 400

5

10

15

20

Re

laxiv

ity (

mM

-1 s

-1)

Shell thickness (nm)

133

significance level), verifying extremely low toxicity levels under biological conditions.

Differential interference contrast (DIC), reflectance (emission and excitation at 640 nm;

Figure 4.14B) and confocal (Figure 4.14C) imaging were used to investigate cellular

nanoparticle internalization. Clear diffraction-limited dark spots corresponding to Gd-NM-

PEG complexes can be observed, verifying that the nanoparticles are internalized within the

cell (Figure 4.14B). Z-series stacks of confocal images were obtained at various depths of

field. Figure 4.14C corresponds to a single image slice near to the center of the cell, clearly

showing that the Gd-NM-PEG (red) are internalized within the cell. The Gd-NM-PEG

accumulates in high concentration in the cytoplasm close to the nucleus, and no significant

morphological changes in the cells were observed.

Higher T1-weighted MRI signal enhancement was found in the sample of RAW 267.4

macrophage cells in PBS treated with Gd-NM-PEG (Figure 4.14F) compared to cells without

Gd-NM-PEG (Figure 4.14E) and buffer reference (Figure 4.14D). In addition, phantoms,

which have been shown increasingly importance for standardizing and optimizing scanner

performance, were prepared using agarose (Figure 4.15). The main idea was to determine the

minimal concentration of Gd-NM-PEG necessary for MR contrast in an environment more

likely in vivo tissues. As can be seen, the MRI contrast increases as the Gd-NM-PEG

concentration increases. Also, phantoms containing 2.3x109 nanoparticles, which corresponds

to a Gd(III) concentration of 10 µmol L-1

, was enough to produce a substantial contrast.

134

Figure 4.14 - (A) Flow cytometry toxicity analysis using SYTOX® red dead cell stain of RAW 264.7 cells after

24 h of incubation with Gd-NM-PEG (green) and Gd-coated-NM (blue). The percentages were

calculated considering the control as 100%. (B) Bright field microscopy and (C) merged confocal

and reflectance images of RAW 264.7 macrophage cells showing the nuclei (blue) and Gd-NM-

PEG (red). T1-weighted MR images of (D) reference (phosphate buffer solution (1x PBS)), (E)

RAW 267.4 macrophage cells in PBS, and (F) RAW 267.4 macrophage cells with Gd-NM-PEG

in PBS. The cell concentration was ~5.2x107cells/mL.

Source: By the author.

Figure 4.15 - Dependence of MR relaxation on nanoparticle concentration and Gd(III) concentration in

phantom: T1-weighted MR images of reference (water), agarose, and the equivalent number of

Gd-NM-PEG (100 μL of solution).

Source: By the author.

D E F

0

20

40

60

80

100

Cellu

lar

Via

bili

ty (

%)

0 1.5x109 5.0x109

Gd-NM (particles/mL)

A

*

10 μm

B C

x109 number ofGd-NMH2O Agarose 0.14 0.57 1.14 2.28 4.57

µM Gd(III) conc.0 0 0.6 2.5 5 10 20

135

4.4 CONCLUSIONS

The Gd(III)-encapsulating nanomatryoshkas that we have designed and synthesized

provide a strong MRI T1 enhancement. The relaxivity was enhanced even further by PEG

functionalization, and increased at higher magnetic fields, a very attractive property for state-

of-the-art MRI applications. The retention of the Gd(III) within the interior of the NM is

excellent, unlike the case of functionalizing Gd(III) chelates on the nanoparticle surface. The

enhanced relaxivity is due to longer-range interactions than are responsible for the relaxivities

of molecular Gd(III) chelates, and are well-described by SBM theory. The biocompatibility,

low toxicity, long term stability, and cellular uptake have been demonstrated in this study

using RAW 264.7 macrophage cells. The T1-weighted MR image of Gd-NM internalized

within RAW 264.7 macrophage cells show a substantial contrast. Given the photothermal

properties that have been shown for NM in previous studies, the Gd-NM-PEG have potential

applications as multifunctional agents for both diagnosis and therapy. Most importantly, the

incorporation of Gd(III) into the NM structure may allow the tracking of the particles in vivo

and investigation of their biodistribution, which is essential to develop safer and more

efficient nanomaterials for medical applications.

136

137

5 CONCLUSIONS AND PERSPECTIVES

In this thesis, we have presented the fabrication of multifunctional gold based-

plasmonic nanoparticles for medical applications. We have included new compounds and

functionalizations into these systems, which allowed the combination of their unique optical

property as phototherapeutic actuator with activated chemotherapy or diagnostic properties.

Specifically, the research was focused on improving three main aspects of the gold

nanomaterials for photothermal therapy: 1) synthesis of gold nanoparticles with high control

of size, shape, and geometry, 2) specific cytotoxicity and enhance accumulation in tumor

cells, and 3) addition of MRI agents that would allow their in vivo tracking and their use as

diagnostic agents.

First, three types of gold based-plasmonic nanoparticles were synthesized, and the

dependence of the surface plasmon resonance with parameters such as shape, size, and

composition were investigated. All systems presented strong NIR absorption and diameter

size less or around 100 nm, characteristics very relevant for medical applications. Their gold

surface also allows conjugation of polymers and biomolecules. Gold nanorods were

synthesized using the seed-mediated method in presence of CTAB with high control of size

and shape. By increasing their aspect ratio, it was possible to tunable the longitudinal plasmon

resonance wavelength. Gold nanomatryoshkas, consisting in a gold core, an interstitial

nanoscale SiO2 layer, and a thin gold shell layer (Au/SiO2/Au) were synthesized with high

control of the thickness of the interstitial silica layer and gold shell, and, consequently, their

optical properties. Further, a promising Au-coated Au2S plasmonic material was also

investigated. Since the one step synthesis usually used to fabricate these particles has not

enabled a good control of the size, distribution, and composition, we proposed a two step

process, in which Au2S small particles are first synthesized and then covered with a gold

layer. This strategy showed promising results with higher control of morphological

parameters.

Second, we have demonstrated the successful coating of PEG-AuNRs into cellular

membranes vesicles purified from A549 cancer cells and loaded with the anticancer agent -

Lapachone. The NIR irradiation led to disruption of the vesicles and release of the PEG-

AuNRs and -Lapachone, and in vitro studies revealed that this multifunctional noncovalent

platform was capable to deliver therapeutic molecules and plasmonic nanostructures upon

NIR irradiation, resulting in an enhanced cytotoxicity against A549 cancer cells. The NQO1-

specific cytotoxicity of -Lap combined with heat generated by laser irradiation of the AuNRs

138

result in a synergic toxicity specific for cancer cells, and no cytotoxicity was observed in

absence of laser irradiation.

Finally, Gd(III)-encapsulating nanomatryoshkas were synthesized. The retention of

the Gd(III) within the interior of the NM provide several advantages, unlike the case of

functionalizing Gd(III) chelates on the nanoparticle surface. They provide a strong MRI T1

enhancement while maintaining the strong near infrared optical properties useful for

photothermal cancer therapy. The relaxivity was enhanced even further by PEG

functionalization, and increased at higher magnetic fields, a very attractive property for state-

of-the-art MRI applications. The enhanced relaxivity is believed to be due to longer-range

interactions that are responsible for the relaxivities of molecular Gd(III) chelates, and are

described by SBM theory. The biocompatibility, low toxicity, long term stability, and cellular

uptake have been demonstrated in this study using RAW 264.7 macrophage cells. Most

importantly, the incorporation of Gd(III) into the NM structure may allow for tracking of the

particles in vivo and investigation of their biodistribution, which is essential to develop safer

and more efficient nanomaterials for medical applications.

Given the photothermal properties that have been shown for gold nanorods and

nanomatryoshkas, the systems developed here have potential applications as multifunctional

agents for activated drug release and phototherapy, diagnosis and treatment, being strong

candidates for applications in personalized cancer medicine. Further studies would be focus

on the optimization of the parameters of the novel two-step synthesis process of Au-coated

Au2S nanoshells, increasing the loading of the anticancer agent -Lap in the CM--Lap-PEG-

AuNRs structure with a detailed investigation of their in vitro and in vivo photothermal

cytotoxicity mechanism, and in vivo MIR investigation of Gd-NM.

139

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